Site-specific integration and stacking of transgenes in soybean via DNA recombinase mediated cassette exchange

ABSTRACT

A targeting method is described that allows precise cassette replacement at a previously characterized genetic locus. A target DNA construct containing a pair of incompatible FRT sites flanking a target cassette was introduced into soybean by regular biolistic transformation. Transgenic events containing a single complete copy of the target site were then selected and retransformed with a donor DNA construct containing the identical pair of incompatible FRT sites flanking a donor cassette. Precise DNA cassette exchange happened between the target cassette and the donor cassette via recombinase mediated cassette exchange (RMCE) so that the donor cassette was introduced at the exact genomic site previously occupied by the target cassette. Through repeated RMCE using additional incompatible FRT sites, multiple groups of transgenes can be stacked at the same genomic locus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/634,775, filed Dec. 10, 2009, which claims the benefit of U.S.Provisional Application No. 61/138,995, filed Dec. 19, 2008, both ofwhich are incorporated herein in their entirety by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named424259seqlist.txt, created on Sep. 13, 2012, and having a size of 206 KBand is filed concurrently with the specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to site-specific integration and stacking oftransgenes in soybean.

BACKGROUND OF THE INVENTION

Current transformation methods using Agrobacterium or biolisticbombardment have challenges such as random integration, multipletransgene copies, and unpredicted integration sites. Acting alone orcombined, these challenges could lead to unpredictable expression orsilencing of introduced transgenes. Though homologous recombination canbe explored to address these challenges (Iida and Terada, (2005) PlantMol. Biol. 59:205-219; Wright et al. (2005) Plant J. 44:693-705),site-specific integration (SSI) mediated by DNA recombinase ispractically a more promising approach to eliminate random integration ofunpredictable copies of a transgene by placing single copy transgeneinto a pre-characterized site in plant genome.

Several site-specific DNA recombination systems, such as the Cre/Iox ofbacteriophage P1, the FLP/FRT of Sacchromyces cerevisiae, and the R/RSof Zygosacchromyces rouxii have been used in site-specific geneintegration studies (Groth and Calos, (2003) J. Mol. Biol. 335:667-678;Ow, (2003) Plant Mol. Biol. 48:183-200). A common feature of thesesystems is that each system consists of a single polypeptide recombinaseCre, FLP, or R, and two identical or almost identical palindromicrecognition sites Iox, FRT, or RS. Each recognition site contains ashort asymmetric spacer sequence where DNA strand exchange takes place,flanked on each side by an inverted repeat sequence where thecorresponding recombinase specifically binds. If two recognitions sitesare located in cis on the same DNA molecule, DNA segment flanked by thetwo sites can be excised if the two sites are in the same orientation,or be inverted if the two sites are in opposite orientations. If tworecognitions sites are each located in trans on two different DNAmolecules, a reciprocal translocation can happen between the two linearDNA molecules, or the two molecules can integrate if at least one ofthem is a circular DNA (Groth and Calos, J. Mol. Biol. 335:667-678(2003); Ow, Plant Mol. Biol. 48:183-200 (2003)).

A simple SSI can target DNA into single recombination site previouslyplaced in a plant genome. Improvement of the single site integrationapproach involved transient Cre expression and the use of mutant Ioxsites to recreate two less compatible Iox sites after integration toreduce subsequent excision of the integrated gene in tobacco (Albert etal. (1995) Plant J. 7:649-659; Day at al. (2000) Genes Dev.14:2869-2880). Similar approach was used to produce SSI events in riceby biolistic bombardment transformation method and the transgene wasproven to be stable and consistently expressed over generations(Srivastava and Ow, (2001) Mol. Breed. 8:345-350; Srivastava et al.(2004) Plant Biotechnol. J. 2:169-179). Using Agrobacterium T-DNA fordonor DNA delivery and a promoter trap to activate selectable markergene and to displace Cre expression upon DNA recombination, ˜2% singleIox site SSI was achieved in Arabidopsis (Vergunst et al. (1998) NucleicAcids Res. 26:2729-2734). The process of SSI is basically irreversibleand thus the genomic site can not be recovered for repeated use.Additionally, since SSI will integrate the entire circular DNA, unwantedcomponents such as the vector backbone is also integrated unless theintegration DNA can be circulated by Cre recombinase to remove unwantedDNA prior to SSI (Srivastava et al. (2004) Plant Biotechnol J.2:169-179; Chawla et al. (2006) Plant Biotechnol. J. 4:209-218; Vergunstet al. (1998) Nucleic Acids Res. 26:2729-2734). To achieve marker-freesite-specific gene integration, a two-step approach was proposed tocombine gene integration using one recombinase system such as Cre/Ioxfollowed by gene excision using another system such as FLP/FRT that isalso conditionally controlled by an inducible promoter (Srivastava andOw, (2004) Trends Biotech. 22:627-629).

If two incompatible recognition sites, which are similar enough to berecognized by the same recombinase but also different enough to preventDNA recombination from happening between them, are located on a linearDNA molecule, DNA segment between the two sites will not be eitherexcised or inverted. When a circular DNA molecule carrying an identicalpair of the incompatible sites is introduced, the circular DNA canintegrate by the corresponding recombinase at either site on the linearDNA to create a collinear DNA molecule with four recognition sites, twofrom the original linear DNA and two from the circular DNA. DNA excisioncan subsequently happen between any pair of compatible sites and resultin the restoration of the original two DNA molecules or the exchange ofthe intervening DNA segments between the two DNA molecules. The latterprocess termed recombinase mediated cassette exchange (RMCE) can beemployed to integrate transgenes directionally into predefined genomesites (Baer and Bode, (2001) Curr. Opin. Biotechnol. 12:473-480; Trinhand Morrision, (2000) J. Immunol. Methods 244:185-193).

RMCE using two identical but oppositely orientated RS sites resulted indonor cassette exchange into the previously placed target site intobacco (Nanto et al. (2005) Plant Biotechnol. J. 3:203-214). The donorvector containing the R recombinase gene and a third RS site to helpeliminating random integration was delivered by Agrobacteriumtransformation. RMCE utilizing both the Cre/Iox and FLP/FRT systems wasused in animal cell cultures to improve RMCE frequency (Lauth et al.(2002) Nucleic Acids Res. 30:e115). RMCE using two directionalincompatible FRT sites was used in Drosophila to achieve cassetteexchange by transiently expressed FLP recombinase between a target DNApreviously placed in the genome and a donor introduced as a circular DNA(Horn and Handler, (2005) Proc. Natl. Acad. Sci. 102:12483-12488). Acomplex gene conversion approach involving Cre/Iox and FLP/FRT mediatedsite-specific integration, RMCE, and homologous recombination wasexplored in maize (Djukanovic et al. (2006) Plant Biotechnol. J.4:345-357).

SUMMARY OF THE INVENTION

The present invention includes:

In one embodiment, a method for stacking multiple expression cassettesof interest into a specific chromosomal site in a soybean genome, saidmethod comprising: (a) transforming a first soybean cell with anisolated nucleic acid fragment comprising at least one first expressioncassette of interest adjacent to a target site, wherein said target sitecomprises a first selectable marker protein-coding sequence, wherein thefirst selectable marker protein-coding sequence is bounded by a firstrecombination site and a second non-identical recombination site; (b)regenerating a transgenic plant from the transformed soybean cell ofstep (a); (c) introducing into a second soybean cell from the transgenicplant of step (b) a transfer cassette, wherein said transfer cassettecomprises a second selectable marker protein-coding sequence, whereinthe second selectable marker protein-coding sequence is bounded by thefirst recombination site and the second non-identical recombinationsites of the target site, and further wherein the transfer cassettefurther comprises at least one second expression cassette of interest,wherein the at least one second expression cassette of interest isbounded by the second selectable marker protein-coding sequence and thesecond non-identical recombination site; and (d) providing a recombinasethat recognizes and implements recombination at the non-identicalrecombination sites.

In another embodiment, the transfer cassette of the method may furthercomprise a third non-identical recombination site bounded by the secondselectable marker protein-coding sequence and the at least one secondexpression cassette of interest.

In another embodiment, the method may further comprise between steps (b)and (c), identifying a transgenic plant of step (b), wherein thetransgenic plant has desirable levels of gene expression for the atleast one first expression cassette of interest.

In another embodiment, the non-identical recombination sites of themethod are selected from the group consisting of FRT1 (SEQ ID NO:50),FRT5 (SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ ID NO:53) and FRT87(SEQ ID NO:54).

In another embodiment, the soybean cell of step (a) is transformed withthe isolated nucleic acid fragment by gene bombardment and the transfercassette of step (c) is introduced into the soybean cell by genebombardment.

In another embodiment, providing of said recombinase in step (d)comprises transiently expressing within said soybean cell an expressioncassette comprising a polynucleotide encoding said recombinase. Therecombinase may be FLP. The FLP recombinase may be synthesized usingmaize preferred codons.

In another embodiment, the first selectable marker protein-codingsequence of the method encodes a protein selected from the groupconsisting of a hygromycin phosphotransferase, a sulfonylurea-tolerantacetolactate synthase, and a sulfonylurea-tolerant acetolactate synthasethat has an amino acid sequence comprising SEQ ID NO:63 or SEQ ID NO:64.

In another embodiment, the target site of the method comprises apromoter operably linked to the first selectable marker protein-codingsequence, and the first recombination site is between the promoter andthe first selectable marker protein-coding sequence.

In another embodiment, a soybean cell, plant or seed having stablyincorporated in its genome a transfer cassette comprising at least threenon-identical recombination sites, where the transfer cassette comprisesa polynucleotide encoding a selectable marker protein-coding sequencebounded by a first recombination site and a second non-identicalrecombination site, wherein the transfer cassette further comprises athird non-identical recombination site bounded by the selectable markerprotein-coding sequence and the second non-identical recombination site,wherein the transfer cassette further comprises at least one expressioncassette of interest, wherein the at least one expression cassette ofinterest is bounded by the third non-identical recombination site andthe second non-identical recombination site. The non-identical FRTrecombination sites may be selected from the group consisting of FRT1(SEQ ID NO:50), FRT5 (SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ IDNO:53) and FRT87 (SEQ ID NO:54). The transfer cassette ma be geneticallylinked to a chromosomal region comprising a sequence selected from thegroup consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62.

In another embodiment, a method for stacking of multiple expressioncassettes of interest into a specific chromosomal site in a soybeangenome, said method comprising: (a) obtaining a transgenic soybean cellcomprising a target site, wherein said target site comprises a firstselectable marker protein-coding sequence, wherein the first selectablemarker protein-coding sequence is bounded by a first recombination siteand a second non-identical recombination site; (b) introducing into thetransgenic soybean cell of step (a) a transfer cassette, wherein saidtransfer cassette comprises a second selectable marker protein-codingsequence, wherein the second selectable marker protein-coding sequenceis bounded by the first recombination site and the second non-identicalrecombination site, and further wherein the transfer cassette furthercomprises at least one expression cassette of interest, wherein the atleast one expression cassette of interest is bounded by the secondselectable marker protein-coding sequence and the second non-identicalrecombination site; and (c) providing a recombinase that recognizes andimplements recombination at the non-identical recombination sites. Thetransfer cassette may further comprise a third non-identicalrecombination site bounded by the second selectable marker gene and theat least one expression cassette of interest. The non-identical FRTrecombination sites may be selected from the group consisting of FRT1(SEQ ID NO:50), FRT5 (SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ IDNO:53) and FRT87 (SEQ ID NO:54). The transfer cassette may begenetically linked to a chromosomal region comprising a sequenceselected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ IDNO:62.

In another embodiment, a method for creating a transgenic soybean cellcomprising a target site suitable for stacking of multiple expressioncassettes of interest into a specific chromosomal site in a soybeangenome, said method comprising transforming a soybean cell with anisolated nucleic acid fragment comprising at least one first expressioncassette of interest adjacent to a target site, wherein said target sitecomprises a selectable marker protein-coding sequence, wherein theselectable marker protein-coding sequence is bounded by a firstrecombination site and a second non-identical recombination site. Thenon-identical recombination sites may be selected from the groupconsisting of FRT1 (SEQ ID NO:50), FRT5 (SEQ ID NO:51), FRT6 (SEQ IDNO:52), FRT12 (SEQ ID NO:53) and FRT87 (SEQ ID NO:54).

In another embodiment, a soybean cell, plant or seed having stablyincorporated in its genome an isolated nucleic acid fragment comprisingat least one first expression cassette of interest adjacent to a targetsite, wherein said target site comprises a selectable markerprotein-coding sequence, wherein the selectable marker protein-codingsequence is bounded by a first recombination site and a secondnon-identical recombination site. The non-identical recombination sitesmay be selected from the group consisting of FRT1 (SEQ ID NO:50), FRT5(SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ ID NO:53) and FRT87 (SEQID NO:54).

In another embodiment, a soybean cell, plant or seed having stablyincorporated in its genome an isolated nucleic acid fragment comprisinga target site, wherein said target site comprises a promoter operablylinked to a selectable marker protein-coding sequence, wherein theselectable marker protein-coding sequence is bounded by a firstrecombination site and a second non-identical recombination site,further wherein the first recombination site is between the promoter andthe selectable marker protein-coding sequence. The target site mayfurther comprise at least one additional non-identical recombinationsite, wherein the at least one additional non-identical recombinationsite is bounded by the selectable marker protein-coding sequence and thesecond non-identical recombination site. The non-identical recombinationsites may be selected from the group consisting of FRT1 (SEQ ID NO:50),FRT5 (SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ ID NO:53) and FRT87(SEQ ID NO:54).

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

The invention can be more fully understood from the following detaileddescription, the accompanying drawings and Sequence Listing which form apart of this application. The Sequence Listing contains the one lettercode for nucleotide sequence characters and the three letter codes foramino acids as defined in conformity with the IUPAC-IUBMB standardsdescribed in Nucleic Acids Research 13:3021-3030 (1985) and in theBiochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the DNA sequence comprising the 4544 bp (base pair)target DNA fragment QC288A. Sequence 109-594 is a synthetic constitutivepromoter SCP1. Sequence 601-673 is the OMEGA 5′ Un-Translated Region(UTR). Sequence 681-728 is a FLP recombinase recognition site FRT1.Sequence 738-1763 is the hygromycin phosphotransferase (hpt) gene codingregion. Sequence 1772-2052 is the nopaline synthase (NOS) terminator.Sequence 2081-3416 is the Arabidopsis ubiquitin 10 gene promoterAT-UBIQ10 PRO including a 5′ UTR intron sequence 3112-3415. Sequence3435-4130 is a yellow fluorescent reporter gene ZS-YELLOW1 N1 (YFP)coding region. Sequence 4136-4402 is another NOS terminator. Sequence4437-4484 is a FLP recombinase recognition site FRT87. See the map ofQC288 in FIG. 2A.

SEQ ID NO:2 is the 7058 bp complete sequence of the target constructQC288 from which SEQ ID NO:1 was isolated as a 4544 bp DNA fragmentQC288A by AscI digestion.

SEQ ID NO:3 is the 8533 bp complete sequence of the donor constructQC329. Sequence 1-48 is a FLP recombinase recognition site FRT1.Sequence 68-2038 is a soybean acetolactate synthase (als) gene codingregion encoding a mutant ALS enzyme insensitive to sulfonylureaherbicides. Sequence 2055-2365 is the potato proteinase II inhibitorgene (PINII) terminator. Sequence 2400-4347 is the soybean ubiquitingene promoter including a 5′ UTR intron 3816-4347. Sequence 4350-5039 isa cyan fluorescent reporter gene AM-CYAN1 (CFP) coding region. Sequence5045-5311 is a NOS terminator. Sequence 5346-5393 is a FLP recombinaserecognition site FRT87. See the map of QC329 in FIG. 2B.

SEQ ID NO:4 is the 6133 bp sequence of the predicted product DNAQC288A329 of RMCE (recombinase mediated cassette exchange) betweenQC288A and QC329. The sequence between the FRT1 and FRT87 sites ofQC288A is replaced by the sequence between the FRT1 and FRT87 sites ofQC329. See the predicted map of QC288A329 in FIG. 3A.

SEQ ID NO:5 is the 4860 bp complete sequence of the FLP recombinaseexpression construct QC292. Sequence 38-523 is the constitutive promoterSCP1. Sequence 530-602 is the OMEGA 5′ UTR. Sequence 617-1888 is a codonoptimized FLP recombinase coding region. Sequence 1895-2204 is the PINIIterminator.

SEQ ID NO:6 is an oligonucleotide that can anneal to SEQ ID NO:7 to makeFRT1 DNA duplex. Restriction enzyme recognition sites are engineered onboth sites of the 48 bp FRT1 sequence for subsequent cloning.

SEQ ID NO:7 is an oligonucleotide complementary to SEQ ID NO:6.

SEQ ID NO:8 is a primer, HSP-F1, specific to a quantitative PCR (qPCR)endogenous control heat shock protein (HSP) gene.

SEQ ID NO:9 is a primer, HSP-R1, specific to a qPCR endogenous controlheat shock protein (HSP) gene.

SEQ ID NO:10 is a VIC labeled MGB fluorescent probe, VIC-MGB, specificto a qPCR endogenous control heat shock protein (HSP) gene.

SEQ ID NO:11 is a primer, 35S-277F, specific to the SCP1 promoter.

SEQ ID NO:12 is a primer, 35S-345R, specific to the SCP1 promoter.

SEQ ID NO:13 is a FAM labeled BHQ1 fluorescent probe 35S-399T specificto the SCP1 promoter for qPCR analysis.

SEQ ID NO:14 is a primer, Hygro-591F, specific to the hpt gene codingregion.

SEQ ID NO:15 is a primer, Hygro-659R, specific to the hpt gene codingregion.

SEQ ID NO:16 is a FAM labeled BHQ1 fluorescent probe, Hygro-612T,specific to the hpt gene coding region.

SEQ ID NO:17 is a primer, Yfp-67F, specific to the YFP gene codingregion.

SEQ ID NO:18 is a primer, Yfp-130R, specific to the YFP gene codingregion.

SEQ ID NO:19 is a FAM labeled BHQ1 fluorescent probe, Yfp-88T, specificto the YFP gene coding region.

SEQ ID NO:20 is a primer, Cfp-F, specific to the CFP gene coding region.

SEQ ID NO:21 is a primer, Cfp-R, specific to the CFP gene coding region.

SEQ ID NO:22 is a FAM labeled MGB fluorescent probe, Cfp-T, specific tothe CFP gene coding region.

SEQ ID NO:23 is a primer, Scp1-S, specific to the SCP1 promoter.

SEQ ID NO:24 is a primer, Hygro-A, specific to the hpt gene codingregion.

SEQ ID NO:25 is a primer, Yfp-3, specific to the YFP gene coding region.

SEQ ID NO:26 is a primer, Frt87-A, specific to a part of the FRT87 siteand its downstream sequence in DNA constructs QC288 and QC329.

SEQ ID NO:27 is a primer, Als-3, specific to the als gene coding region.

SEQ ID NO:28 is a primer, Hpt-1, specific to the hpt gene coding region.

SEQ ID NO:29 is a primer, Hygro-2, specific to the hpt gene codingregion.

SEQ ID NO:30 is a primer, Yfp-1, specific to the YFP gene coding region.

SEQ ID NO:31 is a primer, Yfp-2, specific to the YFP gene coding region.

SEQ ID NO:32 is a primer, Cyan-1, specific to the CFP gene codingregion.

SEQ ID NO:33 is a primer, Cyan-2, specific to the CFP gene codingregion.

SEQ ID NO:34 is a primer, PinII-100R, specific to the PINII terminator.

SEQ ID NO:35 is a primer, PinII-2F, specific to the PINII terminator.

SEQ ID NO:36 is a primer, Vec81, specific to the vector backbone ofconstructs QC288, QC292, and QC329.

SEQ ID NO:37 is a primer, Flp-A, specific to the FLP gene coding region.

SEQ ID NO:38 is an oligonucleotide that can anneal to SEQ ID NO:39 tomake FRT12 DNA duplex. Restriction enzyme recognition sites areengineered on both sites of the 48 bp FRT12 sequence for subsequentcloning.

SEQ ID NO:39 is an oligonucleotide complementary to SEQ ID NO:38.

SEQ ID NO:40 is an oligonucleotide that can anneal to SEQ ID NO:41 tomake FRT6 DNA duplex. Restriction enzyme recognition sites areengineered on both sites of the 48 bp FRT6 sequence for subsequentcloning.

SEQ ID NO:41 is an oligonucleotide complementary to SEQ ID NO:40.

SEQ ID NO:42 is the DNA sequence comprising a 5490 bp basic donorconstruct QC422 for transgene stacking. Sequence 1-48 is a FLPrecombinase recognition site FRT1. Sequence 68-2038 is the soybeanacetolactate synthase (als) gene coding region encoding a mutant ALSenzyme insensitive to sulfonylurea herbicides. Sequence 2055-2365 is thepotato proteinase II inhibitor gene (PINII) terminator. Sequence2422-2469 is a FLP recombinase recognition site FRT12. Sequence2510-2557 is another FLP recombinase recognition site FRT87. Multiplerestriction enzyme recognition sites are engineered between the FRT12and FRT87 sites for the insertion of trait genes. See the map of QC422in FIG. 11A.

SEQ ID NO:43 is the DNA sequence comprising a 4372 bp basic donorconstruct QC429 for transgene stacking. Sequence 1-48 is a FLPrecombinase recognition site FRT1. Sequence 58-1083 is the hygromycinphosphotransferase (hpt) gene coding region. Sequence 1092-1372 is thenopaline synthase (NOS) terminator. Sequence 1400-1447 is a FLPrecombinase recognition site FRT12. Multiple restriction enzymerecognition sites are engineered upstream of the FRT12 site for theinsertion of trait genes. See the map of QC429 in FIG. 11B.

SEQ ID NO:44 is the DNA sequence comprising a 4444 bp basic donorconstruct QC459 for transgene stacking. Sequence 1-48 is a FLPrecombinase recognition site FRT1. Sequence 58-1083 is the hygromycinphosphotransferase (hpt) gene coding region. Sequence 1092-1372 is thenopaline synthase (NOS) terminator. Sequence 1400-1447 is a FLPrecombinase recognition site FRT6. Sequence 1472-1519 is another FLPrecombinase recognition site FRT87. Multiple restriction enzymerecognition sites are engineered between the FRT6 and FRT87 sites forthe insertion of trait genes. See the map of QC459 in FIG. 13A.

SEQ ID NO:45 is the DNA sequence comprising a 5394 bp basic donorconstruct QC428 for transgene stacking. Sequence 1-48 is a FLPrecombinase recognition site FRT1. Sequence 68-2038 is the soybeanacetolactate synthase (als) gene coding region encoding a mutant ALSenzyme insensitive to sulfonylurea herbicides. Sequence 2055-2365 is thepotato proteinase II inhibitor gene (PINII) terminator. Sequence2422-2469 is a FLP recombinase recognition site FRT6. Multiplerestriction enzyme recognition sites are engineered upstream of the FRT6site for the insertion of trait genes. See the map of QC428 in FIG. 13B.

SEQ ID NO:46 is the predicted QC288A422 DNA resulted from a RMCE betweenQC288A and QC422. Sequence 109-594 is a synthetic constitutive promoterSCP1. Sequence 601-673 is the OMEGA 5′ Un-Translated Region (UTR).Sequence 681-728 is a FLP recombinase recognition site FRT1. Sequence748-2178 is the soybean acetolactate synthase (als) gene coding regionencoding a mutant ALS enzyme insensitive to sulfonylurea herbicides.Sequence 2735-3045 is the potato proteinase II inhibitor gene (PINII)terminator. Sequence 3102-3149 is a FLP recombinase recognition siteFRT12. Sequence 3190-3237 is a FLP recombinase recognition site FRT87.The sequences of group 1 transgenes which can be any trait genes ofchoice are not included.

SEQ ID NO:47 is the predicted QC288A422-429 DNA resulted from a RMCEbetween QC288A422 and QC429. Sequence 109-594 is a syntheticconstitutive promoter SCP1. Sequence 601-673 is the OMEGA 5′Un-Translated Region (UTR). Sequence 681-728 is a FLP recombinaserecognition site FRT1. Sequence 738-1763 is the hygromycinphosphotransferase (hpt) gene coding region. Sequence 1772-2052 is thenopaline synthase (NOS) terminator. Sequence 2080-2127 is a FLPrecombinase recognition site FRT12. Sequence 2168-2215 is a FLPrecombinase recognition site FRT87. The sequences of group 1 and group 2transgenes which can be any trait genes of choice are not included.

SEQ ID NO:48 is the predicted QC288A422-459 DNA resulted from a RMCEbetween QC288A422 and QC459. Sequence 109-594 is a syntheticconstitutive promoter SCP1. Sequence 601-673 is the OMEGA 5′Un-Translated Region (UTR). Sequence 681-728 is a FLP recombinaserecognition site FRT1. Sequence 738-1763 is the hygromycinphosphotransferase (hpt) gene coding region. Sequence 1772-2052 is thenopaline synthase (NOS) terminator. Sequence 2080-2127 is a FLPrecombinase recognition site FRT6. Sequence 2152-2199 is a FLPrecombinase recognition site FRT12. Sequence 2240-2287 is a FLPrecombinase recognition site FRT87. The sequences of group 1 and group 2transgenes which can be any trait genes of choice are not included.

SEQ ID NO:49 is the predicted QC288A422-459-460 DNA resulted from a RMCEbetween QC288A422-459 and QC428. Sequence 109-594 is a syntheticconstitutive promoter SCP1. Sequence 601-673 is the OMEGA 5′Un-Translated Region (UTR). Sequence 681-728 is a FLP recombinaserecognition site FRT1. Sequence 748-2178 is the soybean acetolactatesynthase (als) gene coding region encoding a mutant ALS enzymeinsensitive to sulfonylurea herbicides. Sequence 2735-3045 is the potatoproteinase II inhibitor gene (PINII) terminator. Sequence 3102-3149 is aFLP recombinase recognition site FRT6. Sequence 3174-3221 is a FLPrecombinase recognition site FRT12. Sequence 3262-3309 is a FLPrecombinase recognition site FRT87. The sequences of group 1, group 2,and group 3 transgenes which can be any trait genes of choice are notincluded.

SEQ ID NO:50 is the nucleotide sequence of the minimal wild-type FRTrecombination site, designated FRT1.

SEQ ID NO:51 is the nucleotide sequence of the minimal FRT5 mutantrecombination site.

SEQ ID NO:52 is the nucleotide sequence of the minimal FRT6 mutantrecombination site.

SEQ ID NO:53 is the nucleotide sequence of the minimal FRT12 mutantrecombination site.

SEQ ID NO:54 is the nucleotide sequence of the minimal FRT87 mutantrecombination site.

SEQ ID NO:55 is 601 nucleotides of 5′ genomic sequence from target line4729.5.1, also called the “A” line.

SEQ ID NO:56 is 2588 nucleotides of 3′ genomic sequence from the targetline 4729.1.

SEQ ID NO:57 is 984 nucleotides of 5′ genomic sequence from the targetline 4729.5.2, also called the “B” line.

SEQ ID NO:58 is 1305 nucleotides of 3′ genomic sequence from the targetline 4729.5.2.

SEQ ID NO:59 is 452 nucleotides of 5′ genomic sequence from the targetline 4729.7.1, also called the “N” line.

SEQ ID NO:60 is 377 nucleotides of 3′ genomic sequence from the targetline 4729.7.1.

SEQ ID NO:61 is 496 nucleotides of 5′ genomic sequence from the targetline 4730.3.1, also called the “C” line.

SEQ ID NO:62 is 543 nucleotides of 3′ genomic sequence from the targetline 4730.3.1.

SEQ ID NO:63 is the amino acid sequence of a herbicide-tolerant soybeanALS protein.

SEQ ID NO:64 is the amino acid sequence of a herbicide-tolerantArabidopsis ALS protein.

SEQ ID NO:65 is the nucleotide sequence of the adaptor-specific primerAP1 used to amplify genomic DNA sequence bordering the transgenic regionof a target line.

SEQ ID NO:66 is the nucleotide sequence of the QC288A-specific primer,Scp1-A, used to amplify 5′ genomic DNA sequence bordering the transgenicregion of a target line.

SEQ ID NO:67 is the nucleotide sequence of the QC288A-specific primer,Vec-S1, used to amplify 3′ genomic DNA sequence bordering the transgenicregion of a target line.

SEQ ID NO:68 is the nucleotide sequence of the adaptor-specific primerAP2 used to amplify genomic DNA sequence bordering the transgenic regionof a target line.

SEQ ID NO:69 is the nucleotide sequence of the QC288A-specific primer,Scp1-A4, used to amplify 5′ genomic DNA sequence bordering thetransgenic region of a target line.

SEQ ID NO:70 is the nucleotide sequence of the QC288A-specific primer,Vec-S2, used to amplify 3′ genomic DNA sequence bordering the transgenicregion of a target line.

SEQ ID NO:71 is the nucleotide sequence of the 288A-1F primer used forRMCE-specific qPCR.

SEQ ID NO:72 is the nucleotide sequence of the Als-163R primer used forRMCE-specific qPCR.

SEQ ID NO:73 is the nucleotide sequence of the FAM-labeled BHQ1 probeAls-110T.

SEQ ID NO:74 is the nucleotide sequence of the Hygro-116R primer usedfor Target-specific qPCR.

SEQ ID NO:75 is the nucleotide sequence of the FAM-labeled BHQ1 probeHygro-79T.

SEQ ID NO:76 is the nucleotide sequence of the 329-1F primer used fordonor-specific qPCR.

SEQ ID NO:77 is the nucleotide sequence of the QC292 primer used forqPCR assay of the Flp construct QC292.

SEQ ID NO:78 is the nucleotide sequence of the Flp-A primer used forqPCR assay of the Flp construct QC292.

SEQ ID NO:79 is the nucleotide sequence of the FAM-labeled BHQ1 probeOMEGA5UTR-87T used for qPCR assay of the Flp construct QC292.

SEQ ID NO:80 is the nucleotide sequence of the 5′ bordersequence-specific sense primer 53-1S1 used for PCR analysis of targetline A.

SEQ ID NO:81 is the nucleotide sequence of the 5′ bordersequence-specific sense primer 70-1S used for PCR analysis of targetline B.

SEQ ID NO:82 is the nucleotide sequence of the 5′ bordersequence-specific sense primer 8H-ScaS1 used for PCR analysis of targetline C.

SEQ ID NO:83 is the nucleotide sequence of the common sense primerCyan-1 used for RMCE 3′ border-specific PCR.

SEQ ID NO:84 is the nucleotide sequence of the 3′ bordersequence-specific antisense primer 53-1A used for PCR analysis of targetline A.

SEQ ID NO:85 is the nucleotide sequence of the 3′ bordersequence-specific antisense primer 70-1A used for PCR analysis of targetline B.

SEQ ID NO:86 is the nucleotide sequence of the 3′ bordersequence-specific antisense primer 8H-VecA used for PCR analysis oftarget line C.

SEQ ID NO:87 is the nucleotide sequence of ORFSTOP-A which contains stopcodons in all open reading frames.

SEQ ID NO:88 is the nucleotide sequence of ORFSTOP-B which contains stopcodons in all open reading frames.

SEQ ID NO:89 is the nucleotide sequence of excision product QC288ME. SEQID NO:90 is the nucleotide sequence of vector QC448.

SEQ ID NO:91 is the nucleotide sequence of vector QC449.

SEQ ID NO:92 is the nucleotide sequence of vector QC477.

SEQ ID NO:93 is the nucleotide sequence of vector QC478.

SEQ ID NO:94 is the nucleotide sequence of vector QC479.

SEQ ID NO:95 is the predicted 8910 bp QC288A436A DNA resulting from aRMCE between QC288A329A and QC436. All components derived from QC329 inQC288A329A are exchanged with components from the donor DNA QC436.Sequence 109-594 is a synthetic constitutive promoter SCP1. Sequence601-673 is the OMEGA 5′ Un-Translated Region (UTR). Sequence 681-728 isa FLP recombinase recognition site FRT1. Sequence 748-1763 is thehygromycin phosphotranserase gene (HPT). Sequence 1772-2052 is thenopaline synthase gene terminator (NOS TERM). Sequence 2080-2127 is aFLP recombinase recognition site FRT12. Sequence 2147-4233 is thesoybean Kunitz proteinase inhibitor gene promoter (KTI3 PRO). Sequence4256-5291 is a fragment of soybean FAD2 desaturase gene (FAD2-1 (TR1)).Sequence 5302-6012 is a fragment of soybean thioesterase gene (TE2(TR4)). Sequence 6022-6481 is a fragment of soybean thioesterase gene(TE2 (TR5)). Sequence 6502-7212 is an inverted copy of the soybeanthioesterase gene fragment (TE2 (TR4)). Sequence 7223-8258 is aninverted copy of the soybean FAD2 desaturase gene fragment (FAD2-1(TR1)). Sequence 8277-8478 is the soybean Kunitz proteinase inhibitorgene terminator (KTI3 TERM). Sequence 8488-8755 is the soybean albumingene terminator (ALB TERM). Sequence 8803-8850 is a FLP recombinaserecognition site FRT87.

SEQ ID NO:96 is the predicted 21727 bp QC288A436A438A DNA resulting froma RMCE between QC288A436A and QC438. All components derived from QC436in QC288A436A are retained and components from the donor DNA QC438 arestacked. Sequence 109-594 is a synthetic constitutive promoter SCP1.Sequence 601-673 is the OMEGA 5′ Un-Translated Region (UTR). Sequence681-728 is a FLP recombinase recognition site FRT1. Sequence 748-2718 isthe mutant soybean acetolactate synthase gene (ALS). Sequence 2735-3045is the potato proteinase II inhibitor gene terminator (PINII TERM).Sequence 3096-3861 is the pea legumin gene terminator (PS-LEG TERM).Sequence 3866-5443 is the Yarrowia diacylglycerol acyltransferase gene(YL-DGAT1). Sequence 5455-6150 is the soybean glycinin 1 gene promoter(GY1 PRO). Sequence 6190-6799 is the soybean beta conglycinin genepromoter (B-CONGLYCININ PRO). Sequence 6815-6919 is the soybean lectinsignal peptide (LECTIN SP). Sequence 6920-7123 is the barley high lysineprotein gene (BHL8). Sequence 7126-8290 is the French bean phaseolingene terminator (PHASEOLIN TERM). Sequence 8322-9349 is the soybeanalbumin gene promoter (ALB PRO). Sequence 9352-9516 is the soybeanribulose-1,5-bisphosphate carboxylase small subunit transit peptide (SSUTP). Sequence 9517-10422 is the Corynebacterium glutamicumdihydrodipicolinate synthetase gene (CORYNE DAP A). Sequence 10432-10956is a soybean MYB2 gene terminator (MYB2 TERM). Sequence 10974-11976 isthe Arabidopsis ubiquitin gene promoter (UBIQ10 PRO). Sequence11977-12280 is an intron of the Arabidopsis ubiquitin gene promoter(AT-UBQ10 INTRON). Sequence 12298-12462 is the soybeanribulose-1,5-bisphosphate carboxylase small subunit transit peptide (SSUTP). Sequence 12463-13644 is a soybean cysteine synthase gene fragment(CGS (TR1)). Sequence 13647-14811 is the French bean phaseolin geneterminator (PHASEOLIN TERM). Sequence 14897-14944 is a FLP recombinaserecognition site FRT12. Sequence 14964-17050 is the soybean Kunitzproteinase inhibitor gene promoter (KTI3 PRO). Sequence 17073-18108 is afragment of soybean FAD2 desaturase gene (FAD2-1 (TR1)). Sequence18119-18829 is a fragment of soybean thioesterase gene (TE2 (TR4)).Sequence 18839-19298 is a fragment of soybean thioesterase gene (TE2(TR5)). Sequence 19319-20029 is an inverted copy of the soybeanthioesterase gene fragment (TE2 (TR4)). Sequence 20040-21075 is aninverted copy of the soybean FAD2 desaturase gene fragment (FAD2-1(TR1)). Sequence 21094-21295 is the soybean Kunitz proteinase inhibitorgene terminator (KTI3 TERM). Sequence 21305-21572 is the soybean albumingene terminator (ALB TERM). Sequence 21620-21667 is a FLP recombinaserecognition site FRT87.

FIG. 1A-1E are schematic descriptions of DNA constructs, relative PCRprimer and Southern probe positions. FIG. 1A: Target DNA fragment QC288Acontains a constitutive promoter scp1 driving the hpt gene fortransformation selection. A FRT1 site (solid triangle) is placed betweenthe scp1 promoter and the hpt coding sequence, a FRT87 (open triangle)site is placed at the 3′ end. A fluorescent reporter gene yfp driven byan Arabidopsis ubiquitin gene promoter ubiq10 is included between thetwo FRT sites. FIG. 1B: Donor construct QC329 contains an identical pairof FRT1-FRT87 sites flanking a promoter-less mutated soybeanacetolactate synthase (als) gene, which can give chlorsulfuronresistance if expressed, and a cyan florescent reporter gene cfp drivenby a soybean ubiquitin promoter ubq. If RMCE happens between the targetand donor DNA, the als gene will be linked to the scp1 promoter in thetarget locus to be expressed and only RMCE events can be selected bychlorsulfuron resistance. FIG. 1C: RMCE product DNA QC288A329 has thesame structure as the target DNA QC288A described in FIG. 1A except thatall the components between the FRT1 and FRT87 sites of QC288A arereplaced by the components between the FRT1 and FRT87 sites of the donorDNA QC329 described in FIG. 1B. FIG. 1D: FLP construct QC292 contains aconstitutive scp1 promoter to drive the flp gene expression to make theFLP recombinase needed for RMCE. FIG. 1E: A RMCE PCR positive controlconstruct QC165 is unrelated to RMCE but contains a scp1:als cassettethat is similar to the scp1-FRT1:als cassette in the RMCE DNA QC288A329.Construct-specific PCR primers and expected PCR product sizes, Southernprobes and restriction enzyme recognition sites are depicted.

FIG. 2A-2C show the maps of the target DNA construct QC288, the donorDNA construct QC329 and the FLP expression construct QC292.

FIG. 3A-3D shows the maps of predicted RMCE DNA QC288A329, FRT1 site SSIDNA QC288A329FRT1, FRT87 site SSI DNA QC288A329FRT87, and excisionproduct QC288ME.

FIG. 4A-4E show PCR detection and confirmation of RMCE. FIG. 4A-4D:Seven putative RMCE events at somatic embryo stage were analyzed by PCRwith four sets of primers specific to the target QC288A, RMCE QC288A329,donor QC329, and FLP QC292 DNA, respectively. Plasmid DNA QC288, QC329,QC292, and QC165 in place of QC288A329, were included as positivecontrols. The wt and No DNA lanes are wild type and no template negativecontrols. Positions of the primers and sizes of expected PCR productsare depicted in FIG. 1. Three events A-1, B-2, and N-1 were false eventshaving only the QC288A-specific band. A faint QC329-specific banddetected in event M-1 suggested that M-1 might as a chimeric eventcontain randomly integrated QC329 DNA in some cells. QC288A329-specificband was detected in three events M-2, M-3, and B-1. No QC288A-specificband was detected in events M-2 and M-3 suggesting that they werecomplete RMCE events. A weak QC288A-specific band was detected in eventB-1 suggesting that some cells of this event still contain the originalQC288A DNA. A faint QC292-specific band was detected in event M-3suggesting that this event might contain QC292 DNA in some cells. FIG.4E: The three QC288A329 positive events M-2, M-3, and B-1 were analyzedby PCR with primers Scp1-S (SEQ ID NO:23) and Frt87-A (SEQ ID NO:26) toamplify a 5982 bp band, almost the entire length of predicted QC288A329transgene. Their parent events M and B containing the QC288A transgenewere included as controls since the same primers would amplify a 4393 bpband from QC288A. The expected bands were amplified from all the fiveevents. The wt and No DNA lanes are wild type and no template negativecontrols.

FIG. 5A-5B show Southern confirmation of RMCE. Leaf genomic DNAextracted from the T0 plants of five selected retransformation eventsM-1, M-2, M-3, B-1, and B-2 were digested with NdeI to make a Southernblot that was sequentially hybridized with yfp, and cfp probes. T1plants of their target parents M and B and wild type plant were includedas controls. FIG. 5A: The yfp probe hybridized to the target events M,B, the random integration event M-1, the SSI event M-3, and the falseevent B-2. No yfp band was detected in the two RMCE events M-2 and B-1indicating that the yfp gene had been displaced. FIG. 5B: The cfp probedid not hybridize to the target events M, B, or the false event B-2. Asexpected, the cfp probe hybridized to the random integration event M-1,the SSI event M-3, and the two RMCE events M-2 and B-1. The cfp bands inthe RMCE events M-2 and B-1 are ˜1617 bp smaller than the correspondingyfp bands in their target parents M and B as predicted from QC288A andQC288A329 maps (FIG. 1A; FIG. 1C). In addition to the middle band thatis ˜1617 bp smaller than the corresponding yfp bands in its parent M,the RMCE event M-2 has two extra cfp bands of random sizes.

FIG. 6 shows the analyses of putative RMCE events at the somatic embryostage. Putative RMCE events from the retransformation of the targetlines were selected by their resistance to chlorsulfuron and identifiedby CFP expression. One CFP negative event A3 was included as negativecontrols for subsequent analyses. The events were first screened with aPCR using 35S-277F (SEQ ID NO:11) and Als-3 (SEQ ID NO:27) primerscommon to all three target lines. The events were analyzed byconstruct-specific qPCR to confirm RMCE and to check donor and Flpintegration. Border-specific PCR analyses including RMCE-specific,target-specific, and full length PCR were done using variouscombinations of the 5′ border-specific, 3′ border-specific, andtransgene-specific primers. Expected sizes of the RMCE 5′ end-specific,RMCE 3′ end-specific, Target 5′ end-specific, Target 3′ end-specific,full length Excision, full length Target, and full length RMCE PCR are1117, 1351, 1036, 732, 1307, 5063, and 6652 bp for target line A events;967, 1180, 886, 561, 986, 4742, and 6331 bp for target line B events;and 1018, 1294, 937, 675, 1151, 4907, and 6496 bp for target line Cevents.

FIG. 7A-7E show border-specific PCR confirmation of RMCE at the somaticembryo stage. Two RMCE events A1 (lane 2), A2 (lane 3) and one escapeevent A3 (lane 4) derived from target line A were analyzed by PCR usingcombinations of gene-specific and border-specific primers. Wild-type DNA(wt; lane 5) and no template (H₂O; lane 6) negative controls wereincluded. The size of each PCR band is given in by next to the 1 Kb DNAladder (lanes 1 and 7). FIG. 7A: RMCE 5′ border-specific PCR withprimers 53-181 and Als-3. FIG. 7B: RMCE 3′ border-specific PCR withprimers 53-1A and Cyan-1. FIG. 7C: Target 5′ border-specific PCR withprimers 53-181 and Hygro-A. FIG. 7D: Target 3′ border-specific PCR withprimers Yfp-3 and 53-1A. FIG. 7E: Full-length PCR with twoborder-specific primers 53-1S1 and 53-1A. The same PCR experimentamplified the 1307 bp excision-specific band for events A1 and A2, the5063 bp target-specific band for event A3 but not the expected 6652 bpRMCE-specific band in the presence of the smaller excision band forevents A1 and A2.

FIG. 8 shows PCR and qPCR analyses of selected T0 plants from variousRMCE events. Multiple T0 plants regenerated from three RMCE eventsderived from two target lines A and C were analyzed with the sameconstruct-specific qPCR and border-specific PCR analyses described inFIG. 6.

FIG. 9A-9E show border-specific PCR confirmation of RMCE at the T0 plantstage. Four T0 plants regenerated from event A2 were analyzed byborder-specific PCR analyses with the same primers used in FIGS. 7A-7E.Five T0 plants regenerated from events C2 and C3 were analyzed bysimilar border-specific PCR analyses except using 5′ border-specificprimer 8H-ScaS1 and 3′ border-specific 8H-VecA specific to the targetline C. Target parent DNA A and C, RMCE events somatic embryo DNA A2 andC2, wild-type (wt) and no template (H₂O) were included as controls. Thesize of each PCR band is given in by next to the 1 Kb DNA ladder. FIG.9A: RMCE 5′ border-specific PCR. FIG. 9B: RMCE 3′ border-specific PCR.FIG. 9C: Target 5′ border-specific PCR. FIG. 9D: Target 3′border-specific PCR. FIG. 9E: Full length PCR. The PCR failed to amplifythe expected 6652 bp RMCE-band for the target line A derived hemizygousRMCE event A2 and T0 plants A2-1, A2-2, A2-3, and A2-4 in the presenceof the 1307 bp Excision-specific band. The PCR amplified the 6496 bpRMCE-specific band for target line C derived homologous RMCE events C2,C3 and T0 plants C2-1, C2-2, C2-3, C3-1, and C3-2 in the absence of asmall Excision-specific band.

FIG. 10A and FIG. 10B show an alignment of predicted Target, RMCE, andExcision sequences surrounding the recombination sites. FIG. 10A:Sequences surrounding the 5′ end recombination site. Excision resultingfrom the recombination between FRT1 and FRT87 sites could restore eitherthe FRT87 site or the FRT1 site depending on the crossing-over position.FIG. 10B: Sequences surrounding the 3′ end recombination site. Sequencesoriginated from the donor are capitalized. Sequences different from theTarget sequence in the alignments are underlined. Sequences of thetransgene DNA fragments from the border-specific PCR analyses (FIGS.9A-9E) matched, respectively, the predicted Target, RMCE, or Excisionsequences and are thus not shown.

FIG. 11A and FIG. 11B show the maps of the donor DNA construct QC422which contains three FRT sites designed for the first round of RMCE andthe donor DNA construct QC429 which contains two FRT sites designed forthe second round of RMCE in the approach to stack two groups oftransgenes. The insertion sites of transgene groups are indicated.

FIG. 12A and FIG. 12B show the maps of predicted first round RMCE DNAQC288A422 and predicted second round RMCE DNA QC288A422-429 in theapproach to stack two groups of transgenes. The insertion sites oftransgene groups are indicated.

FIG. 13A and FIG. 13B show the maps of the donor DNA construct QC459which contains three FRT sites designed for the second round of RMCE andthe donor DNA construct QC428 which contains two FRT sites designed forthe third round of RMCE in the approach to stack three groups oftransgenes. The insertion sites of transgene groups are indicated.

FIG. 14A and FIG. 14B show the maps of predicted second round RMCE DNAQC288A422-459 and predicted third round RMCE DNA QC288A422-459-460 inthe approach to stack three groups of transgenes. The insertion sites oftransgene groups are indicated.

FIG. 15A-15C show vectors 00448 and its Gateway versions QC449 andQC449i that are useful for creation of target sites during developmentof trait-containing transgenic product lines. Unique cloning sites XmaI,SmaI, FseI, NotI, and XhoI are labeled. The two AscI sites can be usedto prepare DNA fragments free of the vector backbone. QC449 and QC449i(inverted) are made by inserting the ATTR1/R2Gateway fragment at theSmaI site of QC448.

FIG. 16A-16E show vectors QC477 and its Gateway versions QC478, QC478i,QC479, QC479i that are useful for creation of target sites duringdevelopment of trait-containing transgenic product lines. ORFSTOP-A (SEQID NO:87) and ORFSTOP-B (SEQ ID NO:88) on each end of the sams:alscassette are different short sequences containing stop codons in allopen reading frames. Unique cloning sites XmaI, SmaI, AgeI, PmeI, SpeI,FseI, NotI, and XhoI are labeled. The two AscI sites can be used toprepare DNA fragments free of the vector backbone. The four Gatewayversions are created for easy cloning to link trait genes to thesams:als cassette. The Gateway fragment ATTR1/R2 in QC478, 478i(inverted), or Gateway fragment ATTR3/R4 in QC479, 479i (inverted), isinserted at the PmeI site of QC477.

FIG. 17A-17D are schematic descriptions of donor DNA constructs for genestacking and predicted RMCE products. FIG. 17A: Donor DNA QC436 for thefirst round of SSI. A third recombination site FRT12 is introducedbetween the FRT1 and FRT87 sites. The promoter-less selectable markergene HPT is placed between the FRT1 and FRT12 sites. Inverted repeats ofthe soybean delta9 desaturase gene fragment (GM-FAD2-1 (TR1)) andthioesterase gene fragment (GM-THIOESTERASE 2 (TR4)) controlled by thecommon KTI3 promoter are placed between the FRT12 and FRT87 sites. FIG.17B: Donor DNA 00438 for the second round of SSI. Only two recombinationsites FRT1 and FRTa2 are kept. The promoter-less selectable marker geneGM-ALS and several trait genes controlled by various promoters andterminators are placed between the FRT1 and FRT12 sites. FIG. 17C:Predicted QC288A436 DNA of RMCE involving the FRT1 and FRT87 sitesbetween the target QC288A329 DNA (FIG. 10) and the QC436 donor DNA. Allthe components between the FRT1 and FRT87 sites of QC288A329 arereplaced by the components between the FRT1 and FRT87 sites of the donorDNA QC436. FIG. 17D: Predicted QC288A436A438 DNA of RMCE involving theFRT1 and FRT12 sites between the target QC288A436 DNA (FIG. 17C) and theQC438 donor DNA. The promoter-less HPT gene between the FRT1 and FRT12sites of QC288A436 is replaced by the components between the FRT1 andFRT12 sites of the donor DNA QC438. All the components between the FRT12and FRT87 sites of QC288A436 are retained in QC288A436A438.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

A “target site” comprises a nucleotide sequence flanked by twonon-identical recombination sites. A target site provides a “specificchromosomal site” for stacking multiple expression cassettes ofinterest.

A “transfer cassette” for use with a given target site comprises anucleotide sequence flanked by the same two non-identical recombinationsites present in the corresponding target site. The terms “transfercassette”, “donor cassette” and “targeting cassette” are usedinterchangeably herein.

A target site and a transfer cassette may each comprise more than twonon-identical recombination sites.

A “donor construct” is a recombinant construct that contains a transfercassette. The terms “donor construct” and “donor vector” are usedinterchangeably herein.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. The terms “regulatory sequence” and “regulatoryelement” are used interchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably, and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When the alleles present at a given locus on apair of homologous chromosomes in a diploid plant are the same thatplant is homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ thatplant is heterozygous at that locus. If a transgene is present on one ofa pair of homologous chromosomes in a diploid plant that plant ishemizygous at that locus.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onoverexpression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the overexpressed sequence (see Vaucheret et al.(1998) Plant J. 16:651-659; and Gura, (2000) Nature 404:804-808).

“Selection agent” refers to a compound which is toxic to non-transformedplant cells and which kills non-transformed tissues when it isincorporated in the culture medium in an “effective amount”, i.e., anamount equal to or greater than the minimal amount necessary to killnon-transformed tissues. Cells can be transformed with an appropriategene, such that expression of that transgene confers resistance to thecorresponding selection agent, via de-toxification or another mechanism,so that these cells continue to grow and are subsequently able toregenerate plants. The gene conferring resistance to the selection agentis termed the “selectable marker gene”, “selectable marker” or“resistance gene”. Transgenic cells that lack a functional selectablemarker gene will be killed by the selection agent. Selectable markergenes include genes conferring resistance to herbicidal compounds.Herbicide resistance genes generally code for a modified target proteininsensitive to the herbicide or for an enzyme that degrades ordetoxifies the herbicide in the plant before it can act (DeBlock et al.(1987) EMBO J. 6:2513-2518, DeBlock et al. (1989) Plant Physiol., 91:691-704). For example, resistance to glyphosate or sulfonylureaherbicides has been obtained by using genes coding for mutant versionsof the target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) and acetolactate synthase (ALS), respectively. Resistance toglufosinate ammonium, bromoxynil and 2,4-dichlorophenoxyacetic acid(2,4-D) has been obtained by using bacterial genes encoding aphosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, respectively, which detoxifythe respective herbicide. “Sulfonylurea herbicides” include but are notlimited to chlorsulfuron, rimsulfuron, nicosulfuron, Classic®, andOust®. A specific selection agent may have one or more correspondingselectable marker genes. Likewise, a specific selectable marker gene mayhave one or more corresponding selection agents. It is appreciated byone skilled in the art that a selection agent may not be toxic to allplant species or to all cell types within a given plant. For a plantspecies susceptible to a given selection agent, it is also appreciatedthat resistance cells, tissues or whole plants may be obtainedindependent of the transformation process, e.g., through chemicalmutagenesis of the target gene or gene amplification of the target geneduring tissue culture.

Examples of suitable selection agents, include but are not limited to,cytotoxic agents such as hygromycin, sulfonylurea herbicides such aschlorsulfuron, nicosulfuron and rimsulfuron, and other herbicides whichact by inhibition of the enzyme acetolactate synthase (ALS), glyphosate,bialaphos and phosphinothricin (PPT). It is also possible to usepositive selection marker systems such as phospho-mannose isomerase andsimilar systems which confer positive growth advantage to transgeniccells.

Any regenerable plant tissue can be used in accordance with the presentinvention. Regenerable plant tissue generally refers to tissue which canbe regenerated into a differentiated plant. For example, such tissuescan include calluses and/or somatic embryos derived from whole zygoticembryos, isolated scutella, anthers, inflorescences and leaf andmeristematic tissues.

Many of the problems associated with random gene integration, such asmultiple transgene copies, unknown integration sites, unpredictedtransgene expression, may be overcome by site-specific integrationtransformation. Various position effects influencing the expression ofrandomly integrated transgenes may be eliminated and as a result, theeffects of regulatory elements such as promoters, terminators,enhancers, and insulators on gene expression may be comparativelyanalyzed. Transgene integration sites may also be characterized andselected for different applications prior to retransformation.

The RMCE approach using two incompatible recombination sites for doublecrossover provides a more controlled way for gene targeting. The RMCEapproach employs a transgenic plant which comprises a first sequenceencoding a first recombination site and a second sequence comprising asecond non-identical recombination site. A transfer cassette is thenintroduced into the transgenic plant, wherein the transfer cassettecomprises the same first sequence encoding the same first recombinationsite and the same second sequence comprising the same secondnon-identical recombination site. Recombination is then accomplished bya recombinase that recognizes and implements recombination at thenon-identical recombination sites. An advantage of the directional RMCEis that DNA cassette exchange is reversible so the RMCE product can beused as new target for next round RMCE using additional recombinationsites to successively stack multiple transgenes at the same locus togenerate allelic transgenes. Furthermore, since RMCE places only onecopy of a transgene at a selected locus, only one transgenic event isneeded for each locus. The cost associated with the production,maintenance, and characterization of large numbers of transgenic eventswith the transgene at unpredicted multiple loci can be eliminated.

Recently, single copy RMCE plants were obtained in Arabidopsis from theretransformation of target plants by T-DNA delivery of a donor cassette.Both the target and donor cassettes were flanked by two incompatible Ioxsites in inverted orientation. The Cre recombinase was provided on aco-transformed T-DNA (Louwerse, J. D., et al. (2007) Plant Physiol.145:1282-1293).

To develop FLP/FRT mediated RMCE technology in soybean, we first createdtransgenic target lines containing a hygromycin selection gene flankedby two incompatible FRT sites via biolistic random integrationtransformation. Homozygous target lines were obtained and retransformedwith a donor DNA containing a chlorsulfuron selection gene flanked bythe same pair of FRT sites. A FLP expression DNA construct wasco-bombarded with the donor DNA to transiently provide FLP recombinaserequired for DNA recombination between the target and donor DNAmolecules. RMCE events were produced from multiple target lines andconfirmed at both somatic embryo and plant stages by extensive molecularcharacterizations.

The success of the current invention opens new ways for transgenicproduct development and transgene expression research. Various targetlines can be produced and selected with respect to parameters, such asgene silencing, tissue-specific expression, agronomic performance, etc.and maintained as production target lines to accept transgenes withdifferent expression preferences. By engineering more FRT sites inspecific arrangements in target or donor constructs, multiple genes canbe stacked reversibly at the same genetic locus by repeated RMCE.Integration of large DNA molecules, such as bacterial artificialchromosomes, could be feasible via RMCE which relies only on the FLPrecombinase catalyzed interactions between FRT sites.

Compositions and methods for the directional, targeted integration ofexogenous nucleotides into a transformed soybean plant are provided. Themethods use non-identical recombination sites in a gene targeting systemwhich facilitates directional targeting of desired genes and nucleotidesequences into corresponding recombination sites previously introducedinto the target plant genome.

In the methods of the invention, a nucleotide sequence flanked by twonon-identical recombination sites is introduced into the targetorganism's genome establishing a target site for insertion of nucleotidesequences of interest. Once a stable plant or cultured tissue isestablished a second construct, or nucleotide sequence of interest,flanked by corresponding recombination sites as those flanking thetarget site, is introduced into the stably transformed plant or tissuesin the presence of a recombinase protein. This process results inexchange of the nucleotide sequences between the non-identicalrecombination sites of the target site and the transfer cassette.

It is recognized that the transformed plant may comprise multiple targetsites; i.e., sets of non-identical recombination sites. In this manner,multiple manipulations of the target site in the transformed plant areavailable. By target site in the transformed plant is intended a DNAsequence that has been inserted into the transformed plants genome andcomprises non-identical recombination sites.

The two-micron plasmid found in most naturally occurring strains ofSaccharomyces cerevisiae, encodes a site-specific recombinase thatpromotes an inversion of the DNA between two inverted repeats. Thisinversion plays a central role in plasmid copy-number amplification. Theprotein, designated FLP protein, catalyzes site-specific recombinationevents. The minimal recombination site (FRT) has been defined andcontains two inverted 13-base pair (bp) repeats surrounding anasymmetric 8-bp spacer. The FLP protein cleaves the site at thejunctions of the repeats and the spacer and is covalently linked to theDNA via a 3′ phosphate.

Site specific recombinases like FLP cleave and religate DNA at specifictarget sequences, resulting in a precisely defined recombination betweentwo identical sites. To function, the system needs the recombinationsites and the recombinase. No auxiliary factors are needed. Thus, theentire system can be inserted into and function in plant cells.

The yeast FLP/FRT site specific recombination system has been shown tofunction in plants. Earlier, the system was utilized for excision ofunwanted DNA. See, Lyznik et al. (1993) Nucleic Acid Res. 21:969-975.Subsequently, non-identical FRTs were used for the exchange, targeting,arrangement, insertion and control of expression of nucleotide sequencesinto the plant genome (PCT Publication No. WO1999025821; PCT PublicationNo. WO1999025840; PCT Publication No. WO1999025854; PCT Publication No.1999025855; and PCT Publication No. WO2007011733; the contents of allare herein incorporated by reference).

To practice the methods of the invention, a transformed organism ofinterest, particularly a soybean plant, containing a target siteintegrated into its genome is needed. The target site is characterizedby being flanked by non-identical recombination sites. A targetingcassette is additionally required containing a nucleotide sequenceflanked by corresponding non-identical recombination sites as thosesites contained in the target site of the transformed organism. Arecombinase which recognizes the non-identical recombination sites andcatalyzes site-specific recombination is required.

It is recognized that the recombinase can be provided by any means knownin the art. That is, it can be provided in the organism or plant cell bytransforming the organism with an expression cassette capable ofexpressing the recombinase in the organism, by transient expression; orby providing messenger RNA (mRNA) for the recombinase or the recombinaseprotein.

By “non-identical recombination sites” is intended that the flankingrecombination sites are not identical in sequence and will not recombineor recombination between the non-identical sites will be reducedcompared to recombination between identical sites. That is, one flankingrecombination site may be a FRT site where the second recombination sitemay be a mutated FRT site. The non-identical recombination sites used inthe methods of the invention prevent or greatly suppress recombinationbetween the two flanking recombination sites and excision of thenucleotide sequence contained therein. Accordingly, it is recognizedthat any suitable non-identical recombination sites may be utilized inthe invention, including FRT and mutant FRT sites, FRT and Iox sites,Iox and mutant Iox sites, as well as other recombination sites known inthe art.

Suitable non-identical recombination site implies that in the presenceof active recombinase, excision of sequences between two non-identicalrecombination sites occurs, if at all, with an efficiency considerablylower than the recombinationally-mediated cassette exchange targetingarrangement of nucleotide sequences into the plant genome. Thus,suitable non-identical sites for use in the invention include thosesites where the efficiency of recombination between the sites is low;for example, where the efficiency is less than about 30 to about 50%, inanother embodiment less than about 10 to about 30%, in anotherembodiment less than about 5 to about 10%. As noted above, therecombination sites in the targeting cassette correspond to those in thetarget site of the transformed plant. That is, if the target site of thetransformed plant contains flanking non-identical recombination sites ofFRTA and FRTB, the targeting cassette will contain the same FRTA andFRTB non-identical recombination sites.

Sequences of minimal and larger than minimal non-identical FRTs siteshave been described for the exchange, targeting, arrangement, insertionand control of expression of nucleotide sequences into the plant genome(PCT Publication No. WO1999025821; PCT Publication No. WO1999025840; PCTPublication No. WO1999025854; PCT Publication No. 1999025855; and PCTPublication No. WO2007011733, the contents of all are hereinincorporated by reference).

It is furthermore recognized that the recombinase, which is used in theinvention, will depend upon the recombination sites in the target siteof the transformed plant and the targeting cassette. That is, if FRTsites are utilized, the FLP recombinase will be needed. In the samemanner, where Iox sites are utilized, the Cre recombinase is required.If the non-identical recombination sites comprise both a FRT and a Ioxsite, both the FLP and Cre recombinase will be required in the plantcell.

The FLP recombinase is a protein which catalyzes a site-specificreaction that is involved in amplifying the copy number of the twomicron plasmid of S. cerevisiae during DNA replication. FLP protein hasbeen cloned and expressed. See, for example, Cox (1993) Proc. Natl.Acad. Sci. U.S.A. 80:4223-4227. The FLP recombinase for use in theinvention may be that derived from the genus Saccharomyces. Therecombinase may be synthesized using plant preferred codons for optimumexpression in a plant of interest. See, for example, U.S. applicationSer. No. 08/972,258 filed Nov. 18, 1997, entitled “Novel Nucleic AcidSequence Encoding FLP Recombinase”, herein incorporated by reference.The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two Iox sites. The Cre recombinase is known in the art. See, forexample, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J.Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet.22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695-5702. Allof which are herein incorporated by reference. Such Cre sequence mayalso be synthesized using plant preferred codons.

Where appropriate, the nucleotide sequences to be inserted in the plantgenome may be optimized for increased expression in the transformedplant. Where mammalian, yeast, or bacterial genes are used in theinvention, they can be synthesized using plant preferred codons forimproved expression. It is recognized that for expression in monocots,dicot genes can also be synthesized using monocot preferred codons.Methods are available in the art for synthesizing plant preferred genes.See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

The plant preferred codons may be determined from the codons utilizedmore frequently in the proteins expressed in the plant of interest. Itis recognized that monocot or dicot preferred sequences may beconstructed as well as plant preferred sequences for particular plantspecies. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlaket al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328; and Murray etal. (1989) Nucleic Acids Research, 17: 477-498. U.S. Pat. No. 5,380,831;U.S. Pat. No. 5,436,391; and the like, herein incorporated by reference.It is further recognized that all or any part of the gene sequence maybe optimized or synthetic. That is, fully optimized or partiallyoptimized sequences may also be used. Additional sequence modificationsare known to enhance gene expression in a cellular host and can be usedin the invention. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequences,which may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The present invention also encompasses novel FLP recombination targetsites (FRT). The FRT has been identified as a minimal sequencecomprising two 11 base pair inverted repeats, separated by an 8 basespacer, as follows (SEQ ID NO:50; PCT Publication No. WO2007011733):

5′-AGTTCCTATTCTCTAGAAAGTATAGGAACT-3′The domains of the minimal FRT recombination site comprise a pair of 11base pair symmetry elements which are the FLP binding sites (nucleotides1-11 and 20-30 of SEQ ID NO:50); the 8 base pair core, or spacer, region(nucleotides 12-19 of SEQ ID NO:50); and the polypyrimidine tracts(nucleotides 3-14 and nucleotides 16-29 of SEQ ID NO:50). A modified ormutant FRT recombination site can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more alterations which include substitutions, additions, and/ordeletions in one or more of these domains.

The eight base pair spacer is involved in DNA-DNA pairing during strandexchange. The asymmetry of the region determines the direction of sitealignment in the recombination event, which will subsequently lead toeither inversion or excision. Most of the spacer can be mutated withouta loss of function. See, for example, Schlake and Bode (1994)Biochemistry 33:12746-12751, herein incorporated by reference.

Mutant FRT sites are provided for use in the practice of the methods ofthe present invention and have been described in PCT Publication No.WO2007011733, the contents of which are herein incorporated byreference. Such mutant sites may be constructed by PCR-basedmutagenesis. While mutant FRT sites are provided herein, it isrecognized that other mutant FRT sites may be used in the practice ofthe invention. The present invention is not the use of a particular FRTor recombination site, but rather that non-identical recombination sitesor FRT sites can be utilized for targeted insertion and expression ofnucleotide sequences in a plant genome. Thus, other mutant FRT sites canbe constructed and utilized based upon the present disclosure.

As discussed above, bringing genomic DNA containing a target site withnon-identical recombination sites together with a vector containing atransfer cassette with corresponding non-identical recombination sites,in the presence of the recombinase, results in recombination. Thenucleotide sequence of the transfer cassette located between theflanking recombination sites is exchanged with the nucleotide sequenceof the target site located between the flanking recombination sites. Inthis manner, nucleotide sequences of interest may be preciselyincorporated into the genome of the host.

It is recognized that many variations of the invention can be practiced.For example, target sites can be constructed having multiplenon-identical recombination sites. Thus, multiple genes or nucleotidesequences can be stacked or ordered at precise locations in the plantgenome. Likewise, once a target site has been established within thegenome, additional recombination sites may be introduced byincorporating such sites within the nucleotide sequence of the transfercassette and the transfer of the sites to the target sequence. Thus,once a target site has been established, it is possible to subsequentlyadd sites, or alter sites through recombination.

Another variation includes providing a promoter or transcriptioninitiation region operably linked with the target site in an organism.For example, the promoter will be 5′ to the first recombination site. Bytransforming the organism with a transfer cassette comprising a codingregion, expression of the coding region will occur upon integration ofthe transfer cassette into the target site. This embodiment provides fora method to select transformed cells, particularly plant cells, byproviding a selectable marker sequence as the coding sequence.

Other advantages of the present system include the ability to reduce thecomplexity of integration of transgenes or transferred DNA in anorganism by utilizing transfer cassettes as discussed above andselecting organisms with simple integration patterns. In the samemanner, preferred sites within the genome can be identified by comparingseveral transformation events. A preferred site within the genomeincludes one that does not disrupt expression of essential sequences andprovides for adequate expression of the transgene sequence.

The methods of the invention also provide for means to combine multiplecassettes at one location within the genome. Recombination sites may beadded or deleted at target sites within the genome.

Any means known in the art for bringing the three components of thesystem together may be used in the invention. For example, a plant canbe stably transformed to harbor the target site in its genome. Using therecombinase, either transiently or stably integrated into the genome ofthe plant, the transfer cassette flanked by corresponding non-identicalrecombination sites is inserted into the transformed plant's genome.

Alternatively, the components of the system may be brought together bysexually crossing transformed plants. In this embodiment, a transformedplant, parent one, containing a target site integrated in its genome canbe sexually crossed with a second plant, parent two, that has beengenetically transformed with a transfer cassette containing flankingnon-identical recombination sites, which correspond to those in plantone. Either plant one or plant two contains within its genome anucleotide sequence expressing recombinase. The recombinase may be underthe control of a constitutive or inducible promoter.

Inducible promoters include heat-inducible promoters,estradiol-responsive promoters, chemical inducible promoters, and thelike. Pathogen inducible promoters include those frompathogenesis-related proteins (PR proteins), which are induced followinginfection by a pathogen; e.g., PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al.(1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) The PlantCell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. In thismanner, expression of recombinase and subsequent activity at therecombination sites can be controlled.

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Promoters which are seed or embryo-specific and may be useful in theinvention include soybean Kunitz trypsin inhibitor (Kti3) (Jofuku andGoldberg, (1989) Plant Cell 1:1079-1093), patatin (potato tubers)(Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin,and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen.Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470;Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein(maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J.7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al.(1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin(bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577),B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al. (1988)EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barleyendosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366),glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J.6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., etal. (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specificgenes operably linked to heterologous coding regions in chimeric geneconstructions maintain their temporal and spatial expression pattern intransgenic plants. Such examples include Arabidopsis thaliana 2S seedstorage protein gene promoter to express enkephalin peptides inArabidopsis and Brassica napus seeds (Vanderkerckhove et al. (1989)Bio/Technology 7:L929-932), bean lectin and bean beta-phaseolinpromoters to express luciferase (Riggs et al. (1989) Plant Sci.63:47-57), and wheat glutenin promoters to express chloramphenicolacetyl transferase (Colot et al. (1987) EMBO J 6:3559-3564).

The compositions and methods of the invention are useful in targetingthe integration of transferred nucleotide sequences to a specificchromosomal site. The nucleotide sequence may encode any nucleotidesequence of interest. Particular genes of interest include those whichprovide a readily analyzable functional feature to the host cell and/ororganism, such as marker genes, as well as other genes that alter thephenotype of the recipient cells, and the like. Thus, genes effectingplant growth, height, susceptibility to disease or insects, nutritionalvalue, and the like may be utilized in the invention. The nucleotidesequence also may encode an antisense sequence to turn off or modifygene expression.

It is recognized that the nucleotide sequences will be utilized in afunctional expression unit or cassette. By functional expression unit orcassette is intended, the nucleotide sequence of interest with afunctional promoter, and in most instances a termination region. Thereare various ways to achieve the functional expression unit within thepractice of the invention. In one embodiment of the invention, thenucleic acid of interest is transferred or inserted into the genome as afunctional expression unit. Alternatively, the nucleotide sequence maybe inserted into a site within the genome which is 3′ to a promoterregion. In this latter instance, the insertion of the coding sequence 3′to the promoter region is such that a functional expression unit isachieved upon integration.

For convenience, for expression in plants, the nucleic acid encodingtarget sites and the transfer cassettes, including the nucleotidesequences of interest, can be contained within expression cassettes. An“expression cassette” will comprise a transcriptional initiation region,or promoter, operably linked to the nucleic acid fragment encoding theRNA of interest. Such an expression cassette may be provided with aplurality of restriction sites for insertion of the gene or genes ofinterest to be under the transcriptional regulation of the regulatoryregions.

The transcriptional initiation region, the promoter, may be native orhomologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign is intended thatthe transcriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

For protein expression, the expression cassette will include in the5-prime to 3-prime direction of transcription, a transcriptional andtranslational initiation region, a DNA sequence of interest, and atranscriptional and translational termination region functional inplants. The termination region may be native with the transcriptionalinitiation region, may be native with the DNA sequence of interest, ormay be derived from another source. Convenient termination regions areavailable from the potato proteinase inhibitor (PinII) gene or fromTi-plasmid of A. tumefaciens, such as the octopine synthase and nopalinesynthase termination regions. See also, Guerineau et al. (1991) Mol.Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon etal. (1991) Genes Dev. 5:141-149; Mogen et al (1990) Plant Cell2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989)Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res.15:9627-9639.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA, 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMVleader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and humanimmunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., andP. Sarnow (1991) Nature, 353:90-94; untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., andGehrke, L., (1987) Nature, 325:622-625; tobacco mosaic virus leader(TMV), (Gallie et al. (1989) Molecular Biology of RNA, pages 237-256,Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273; and maize chloroticmottle virus leader (MCMV) (Lommel, S. A. et al. (1991) Virology,81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology,84:965-968. Other methods known to enhance translation can also beutilized, for example, introns, and the like.

The expression cassettes may contain one or more than one gene ornucleic acid sequence to be transferred and expressed in the transformedplant. Thus, each nucleic acid sequence will be operably linked to 5′and 3′ regulatory sequences. Alternatively, multiple expressioncassettes may be provided.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues.

See generally, G. T. Yarranton (1992) Curr. Opin. Biotech., 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA, 89:6314-6318;Yao et al. (1992) Cell, 71:63-72; W. S. Reznikoff (1992) Mol.Microbiol., 6:2419-2422; Barkley et al. (1980) The Operon, pp. 177-220;Hu et al. (1987) Cell, 48:555-566; Brown et al. (1987) Cell, 49:603-612;Figge et al. (1988) Cell, 52:713-722; Deuschle et al. (1989) Proc. Natl.Acad. Aci. USA, 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad.Sci. USA, 86:2549-2553; Deuschle et al. (1990) Science, 248:480-483; M.Gossen (1993) PhD Thesis, University of Heidelberg; Reines et al. (1993)Proc. Natl. Acad. Sci. USA, 90:1917-1921; Labow et al. (1990) Mol. CellBio., 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA,89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA,88:5072-5076; Wyborski et al. (1991) Nuc. Acids Res., 19:4647-4653; A.Hillenand-Wissman (1989) Topics in Mol. and Struc. Biol., 10:143-162;Degenkolb et al. (1991) Antimicrob. Agents Chemother., 35:1591-1595;Kleinschnidt et al. (1988) Biochemistry, 27:1094-1104; Gatz et al.(1992) Plant J., 2:397-404; A. L. Bonin (1993) PhD Thesis, University ofHeidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA,89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.,36:913-919; Hlavka et al. (1985) Handbook of Exp. Pharmacology, 78; Gillet al. (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The methods of the invention can also be utilized to find optimalintegration sites within a plant genome. In this manner, a plant istransformed with an expression cassette comprising a selectable markergene. The expression cassette is a target site as the marker gene isflanked by non-identical recombination sites. Transformed protoplast,tissues, or whole plants can be tested to determine the levels ofactivity of the inserted gene. By comparison of cellular activities ofthe gene in different insertion sites, preferred integration sites maybe found wherein the gene is expressed at high or acceptable levels.These plants can then be utilized with subsequent retargeting techniquesto replace the marker gene with other genes or nucleotide sequences ofinterest. In the same manner, multiple genes may be inserted at theoptimal site for expression.

Alternatively, the process of creating a target line can be combinedwith the development of a trait-containing transgenic product line. Inthis scheme, a target site will be obtained as a by-product once thetransgenic product line is selected and well characterized. Since atrait gene or a group of genes responsible for the trait is alreadyplaced at a particular locus, that site is convenient for stacking ofadditional new traits through RMCE. A common selectable marker genecassette is usually used for plant transformation to facilitate theselection of transformed events, such as the 35S:hpt and sams:alscassettes used in soybean transformation (US patent publication WO00/37662) and the 35S:BAR and UBIQ:GAT cassettes used in maizetransformation. Consequently, two incompatible recombinase recognitionsites can be incorporated in the selectable marker gene cassette whichcan then be linked to any trait gene of interest for transformation.Once integrated in a plant genome the incorporated incompatiblerecombinase sites can be used for RMCE.

Methods for transformation of plants are known in the art. Suitablemethods of transforming plant cells include microinjection (Crossway etal. (1986) Biotechniques 4:320-334), electroporation (Riggs et al.(1986) Proc. Natl. Acad. Sci. USA, 83:5602-5606), Agrobacterium mediatedtransformation (Hinchee et al. (1988) Biotechnology, 6:915-921), directgene transfer (Paszkowski et al. (1984) EMBO J., 3:2717-2722), andballistic particle acceleration (see, for example, Sanford et al., U.S.Pat. No. 4,945,050; WO91/10725 and McCabe et al. (1988) Biotechnology,6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet.,22:421-477; Sanford et al. (1987) Particulate Science and Technology,5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology, 6:923-926 (soybean);Datta et al. (1990) Biotechnology, 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA, 85:4305-4309 (maize); Klein et al. (1988)Biotechnology, 6:559-563 (maize); WO91/10725 (maize); Klein et al.(1988) Plant Physiol., 91:440-444 (maize); Fromm et al. (1990)Biotechnology, 8:833-839; and Gordon-Kamm et al. (1990) Plant Cell,2:603-618 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature(London), 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci.USA, 84:5345-5349 (Liliaceae); De Wet et al. (1985) In The ExperimentalManipulation of Ovule Tissues, ed. G. P. Chapman et al., pp. 197-209.Longman, N.Y. (pollen); Kaeppler et al. (1990) Plant Cell Reports,9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet., 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell,4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports,12:250-255 and Christou and Ford (1995) Annals of Botany, 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology, 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells which have been transformed may be grown into plants inaccordance with conventional approaches. See, for example, McCormick etal. (1986) Plant Cell Reports, 5:81-84. These regenerated plants maythen be pollinated with either the same transformed strain or differentstrains, and the resulting hybrid having the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that the subject phenotypic characteristic is stably maintainedand inherited and then seeds harvested to ensure the desired phenotypeor other property has been achieved.

Embodiments of the invention include the following:

In one embodiment, a soybean cell, plant or seed having stablyincorporated in its genome an isolated nucleic acid fragment comprisingat least one first expression cassette of interest adjacent to a targetsite, wherein said target site comprises a selectable markerprotein-coding sequence, wherein the selectable marker protein-codingsequence is bounded by a first recombination site and a secondnon-identical recombination site. The target site may be geneticallylinked to a chromosomal region comprising a sequence selected from thegroup consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ ID NO:62.

In another embodiment, a soybean cell, plant or seed having stablyincorporated in its genome an isolated nucleic acid fragment comprisinga target site, wherein said target site comprises a promoter operablylinked to a selectable marker protein-coding sequence, wherein theselectable marker protein-coding sequence is bounded by a firstrecombination site and a second non-identical recombination site,further wherein the first recombination site is between the promoter andthe selectable marker protein-coding sequence. The target site mayfurther comprise at least one additional non-identical recombinationsite, wherein the at least one additional non-identical recombinationsite is bounded by the selectable marker protein-coding sequence and thesecond non-identical recombination site. The target site may begenetically linked to a chromosomal region comprising a sequenceselected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ IDNO:62.

In another embodiment, a soybean cell, plant or seed having stablyincorporated in its genome a transfer cassette comprising at least threenon-identical recombination sites, where the transfer cassette comprisesa polynucleotide encoding a selectable marker protein-coding sequencebounded by a first recombination site and a second non-identicalrecombination site, wherein the transfer cassette further comprises athird non-identical recombination site bounded by the selectable markerprotein-coding sequence and the second non-identical recombination site,wherein the transfer cassette further comprises at least one expressioncassette of interest, wherein the at least one expression cassette ofinterest is bounded by the third non-identical recombination site andthe second non-identical recombination site. The transfer cassette maybe genetically linked to a chromosomal region comprising a sequenceselected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 and SEQ IDNO:62.

In another embodiment, a method for stacking of multiple expressioncassettes of interest into a specific chromosomal site in a soybeangenome, said method comprising: (a) transforming a first soybean cellwith an isolated nucleic acid fragment comprising at least a firstexpression cassette of interest adjacent to a target site, wherein saidtarget site comprises a first selectable marker protein-coding sequence,wherein the first selectable marker protein-coding sequence is boundedby a first recombination site and a second non-identical recombinationsite; (b) regenerating a transgenic plant from the transformed soybeancell of step (a); (c) introducing into a second soybean cell from thetransgenic plant of step (b) a transfer cassette, wherein said transfercassette comprises a second selectable marker protein-coding sequence,wherein the second selectable marker protein-coding sequence is boundedby the first recombination site and the second non-identicalrecombination sites of the target site; and (d) providing a recombinasethat recognizes and implements recombination at the non-identicalrecombination sites. Optionally, the method may further comprise,between steps (b) and (c), identifying a transgenic plant of step (b),wherein the transgenic plant has desirable levels of gene expression forthe at least one first expression cassette of interest.

In another embodiment, a second method for stacking of multipleexpression cassettes of interest into a specific chromosomal site in asoybean genome, said method comprising: (a) obtaining a transgenicsoybean cell comprising a target site, wherein said target sitecomprises a first recombination site and a second non-identicalrecombination site; (b) introducing into the transgenic soybean cell ofstep (a) a transfer cassette, wherein said transfer cassette comprises aselectable marker protein-coding sequence, wherein the selectable markerprotein-coding sequence is bounded by the first recombination site andthe second non-identical recombination site; and (c) providing arecombinase that recognizes and implements recombination at thenon-identical recombination sites.

In another embodiment, a method for creating a transgenic soybean cellcomprising a target site suitable for stacking of multiple expressioncassettes of interest into a specific chromosomal site in a soybeangenome, said method comprising transforming a soybean cell with anisolated nucleic acid fragment comprising at least a first expressioncassette of interest adjacent to a target site, wherein said target sitecomprises a selectable marker protein-coding sequence, wherein theselectable marker protein-coding sequence is bounded by a firstrecombination site and a second non-identical recombination site.

In one or more of the embodiments, the transfer cassette may furthercomprise a third non-identical recombination site bounded by the secondselectable marker protein-coding sequence and the second non-identicalrecombination site.

In one or more of the embodiments, the transfer cassette may furthercomprise at least one second expression cassette of interest, whereinthe at least one second expression cassette of interest is bounded bythe third non-identical recombination site and the second non-identicalrecombination site.

In one or more of the embodiments, the non-identical recombination sitesmay be selected from the group consisting of FRT1 (SEQ ID NO:50), FRT5(SEQ ID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ ID NO:53) and FRT87 (SEQID NO:54).

In one or more of the embodiments, the soybean cell may be transformedwith the isolated nucleic acid fragment by gene bombardment.

In one or more of the embodiments, the transfer cassette may beintroduced into the soybean cell by gene bombardment.

In one or more of the embodiments, providing the recombinase comprisestransiently expressing within the soybean cell an expression cassettecomprising a polynucleotide encoding the recombinase. In anotherembodiment, the recombinase is FLP. In another embodiment, the FLP hasbeen synthesized using maize preferred codons.

In one or more or the embodiments, the first selectable markerprotein-coding sequence encodes a protein selected from the groupconsisting of a hygromycin phosphotransferase and asulfonylurea-tolerant acetolactate synthase. For example, thesulfonylurea-tolerant acetolactate synthase may have an amino acidsequence comprising SEQ ID NO:63 or SEQ ID NO:64.

In one or more of the embodiments, the target site comprises a promoteroperably linked to the first selectable marker protein-coding sequence,wherein the first recombination site is between the promoter and thefirst selectable marker protein-coding sequence.

A recombinant DNA construct of the present invention may comprise atleast one regulatory sequence.

A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs of thepresent invention. The promoters can be selected based on the desiredoutcome, and may include constitutive, tissue-specific, inducible, orother promoters for expression in the host organism.

A composition of the present invention is a plant comprising in itsgenome any of the recombinant DNA constructs of the present invention.Compositions also may include any progeny of the plant, and any seedobtained from the plant or its progeny, wherein the progeny or seedcomprises within its genome the recombinant DNA construct (orsuppression DNA construct). Progeny includes subsequent generationsobtained by self-pollination or out-crossing of a plant.

A method of producing seed (for example, seed that can be sold as atrait-containing product offering) comprising any of the precedingmethods, and further comprising obtaining seeds from said progeny plant,wherein said seeds comprise in their genome said recombinant DNAconstruct.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants may beself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Aspects of the present invention are exemplified in the followingExamples. It should be understood that these Examples, while indicatingembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

In the discussion below, parts and percentages are by weight and degreesare Celsius, unless otherwise stated. Sequences of promoters, cDNA,adaptors, and primers listed herein are in the 5′ to 3′ orientationunless described otherwise. Routine techniques in molecular biology aredescribed in Ausubel et al. Current Protocols in Molecular Biology; JohnWiley & Sons: New York, 1990 and Sambrook et al. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press: Cold SpringHarbor, 1989.

Example 1 FLP/FRT Mediated RMCE Experimental Design and DNA Construction

The target QC288A and donor QC329 constructs were designed eachcontaining a FRT1 site (solid triangle) and a FRT87 site (open triangle)in the same orientation (FIG. 1A, FIG. 2A, FIG. 1B, FIG. 2B). FRT1 isthe wild-type recombination site for FLP recombinase and FRT87 is amodified recombination site (PCT Publication No. WO2007011733 publishedon Jan. 25, 2007). The circular QC329 DNA could integrate into thelinear QC288A DNA previously placed in soybean genome by FLP recombinasemediated DNA recombination at either the FRT1 site or the FRT87 site toform collinear intermediates that contained two FRT1 sites and two FRT87sites. FLP recombinase mediated excision could occur to excise theintervening fragment either between the two FRT1 sites or between theFRT87 sites. The net result of the integration via recombination betweenone identical pair of FRT sites and subsequent excision viarecombination between the other pair of FRT sites would be thereplacement the target DNA with the recombined RMCE DNA QC288A329 (FIG.1C, FIG. 3A). If the gene excision step failed to occur, theintermediates would remain as SSI events containing all the componentsof both the target and donor constructs (FIG. 3B, FIG. 3C).

The target construct QC288A contained a selectable marker gene hptdriven by a constitutive promoter scp1 and transgenic events wereselected with hygromycin. The donor construct QC329 contained apromoter-less selectable marker gene als that would not be expressedunless a promoter was placed in front of it. During retransformation thepromoter-less als gene of QC329 could be brought downstream of the scp1promoter by RMCE and the resulted QC288A329 DNA would enableretransformation events to be selected with chlorsulfuron due to the alsgene activation. SSI events with QC329 integrated at the FRT1 site wouldalso be similarly selected. However, random integration events of QC329would not be able to survive chlorsulfuron selection unless thepromoter-less als gene happened to insert downstream of a nativepromoter. A yellow fluorescent reporter gene cassette ubiq10:yfp wasincluded in QC288A and a cyan fluorescent reporter gene cassette ubq:cfpwas included in QC329 to facilitate transgenic events characterization(FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B).

The target DNA construct QC288 was made through multiple cloning stepsusing components from existing DNA constructs (Li et al, (2007) PlantMol. Biol. 65:329-341). Restriction enzymes and DNA modifying enzymessuch as DNA polymerase Klenow fragment and DNA ligase were usedaccording to manufacturers' recommendations (New England Biolabs,Beverly, Mass., USA; Promega, Madison, Wis., USA; or Invitrogen,Carlsbad, Calif., USA). The FRT87 recombination site DNA fragment wasreleased from construct PHP20234 with BamHI/SmaI digestion and clonedinto BamHI/PvuII sites of pZSL141 to make QC278 consisted of als-FRT87.The FRT1 recombination site was made by annealing two 92 bpcomplementary oligos SEQ ID NO:6 and 7 engineered with multiple cloningsites (Sigma-Genosys, The Woodlands, Tex., USA). The BamHI/HpaI FRT1 DNAfragment was cloned into the BamHI/SmaI sites of construct pZSL90 tomake QC280 consisted of scp1-FRT1:yfp:nos. The DNA fragment containinghpt coding sequence and nos terminator was release from pZSL93 withSpeI/XmaI digestion and cloned into the SpeI/XmaI sites of QC280 to makeQC282 consisted of scp1-FRT1:hpt:nos+yfp:nos. Thescp1-FRT1:hpt:nos+yfp:nos fragment was released from QC282 withHindIII/EcoRV digestion and cloned into the HindIII/BamHI sites of QC278(the BamHI site was completely filled in with Klenow DNA polymerase) tomake QC284 consisted of scp1-FRT1:hpt:nos+yfp:nos-FRT87. The ubiq10promoter fragment was released from construct QC257i with BamHI/XmaIdigestion and cloned into the BamHI/XmaI sites of QC282 to make QC286consisted of scp1-FRT1:hpt:nos+ubiq10:yfp:nos. The final targetconstruct QC288 consisted of scp1-FRT1:hpt:nos+ubiq10:yfp:nos-FRT87 wasmade by cloning BamHI/SphI fragment from QC286 into the BamHI/SphI sitesof QC284 (FIG. 2A).

The donor construct QC329 was made by first cloning the BamHI/HindIIIFRT1 DNA fragment into the BamHI/HindIII sites of construct pSMamCyan tomake QC281 consisted of FRT1:cfp:nos. The FRT87 site was added bycloning the AflII/SpeI fragment of the above QC284 (the SpeI site wascompletely filled with Klenow DNA polymerase) into the AflII/EcoRI sitesof QC281 (the EcoRI site was completely filled in with Klenow DNApolymerase) to make QC283 consisted of FRT1:cfp:nos-FRT87. The ubiq10promoter fragment was released from QC257i with BamHI/XmaI digestion andcloned into the BamHI/XmaI sites of QC283 to make QC285 consisted ofFRT1-ubiq10:cfp:nos-FRT87. Separately, the als:pinII fragment wasreleased from QC257i with BgIII/KpnI digestion (the BgIII site wascompletely filled in with Klenow DNA polymerase) and cloned into theEcoCRI/KpnI sites of pZSL81 to make QC279 consisted of als:pinII. TheSpeI/XmaI fragment of QC279 was then cloned into the SpeI/XmaI sites ofQC285 to make QC287 consisted of FRT1-als:pinII+ubiq10:cfp:nos-FRT87.The ubiq10 promoter was later replaced with a soybean ubiquitin promoterubq by cloning the XmaI/NcoI ubq promoter fragment from QC319 into theXmaI/NcoI sites of QC287 to make the final donor vector QC329 consistedof FRT1-als:pinII+ubq:cfp:nos-FRT87 (FIG. 2B).

The FLP expression construct QC292 containing scp1:flp:pinII was made bysimply cloning the BamHI/HindIII scp1 promoter fragment from pZSL90 intothe BamHI/HindIII sites of construct PHP5096 (FIG. 2C).

Example 2 Target Event Creation and Characterization

The scp1-FRT1:hpt:nos+ubiq10:yfp:nos-FRT87 cassette of QC288 wasreleased as a 4544 bp DNA fragment QC288A with AscI digestion, resolvedby agarose gel electrophoresis, and purified using a Qiagen gelextraction kit (Qiagen, Valencia, Calif., USA). Soybean embryogenicsuspension cultures were transformed with QC288A DNA following thebiolistic bombardment transformation protocol using 30 μg/ml hygromycinfor transgenic events selection (Li et al, (2007) Plant Mol. Biol.65:329-341; Klein et al. (1987) Nature 327:70-73; U.S. Pat. No.4,945,050)).

Soybean somatic embryos from the Jack cultivar were induced as follows.Cotyledons (smaller than 3 mm in length) were dissected fromsurface-sterilized, immature seeds and were cultured for 6-10 weeksunder fluorescent light at 26° C. on a Murashige and Skoog media (“MSmedia”) containing 0.7% agar and supplemented with 10 mg/ml2,4-dichlorophenoxyacetic acid (2,4-D). Globular stage somatic embryos,which produced secondary embryos, were then excised and placed intoflasks containing liquid MS medium supplemented with 2,4-D (10 mg/ml)and cultured in the light on a rotary shaker. After repeated selectionfor clusters of somatic embryos that multiplied as early, globularstaged embryos, the soybean embryogenic suspension cultures weremaintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C.with fluorescent lights on a 16:8 hour day/night schedule. Cultures weresubcultured every two weeks by inoculating approximately 35 mg of tissueinto 35 ml of the same fresh liquid MS medium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment using a DuPont™ Biolistic™ PDS1000/HEinstrument (helium retrofit) (Bio-Rad Laboratories, Hercules, Calif.).To 50 μl of a 60 mg/ml 1.0 mm gold particle suspension were added (inorder): 30 μl of 10 ng/μl QC288A DNA fragment, 20 μl of 0.1 Mspermidine, and 25 μl of 5 M CaCl₂. The particle preparation was thenagitated for 3 minutes, spun in a centrifuge for 10 seconds and thesupernatant removed. The DNA-coated particles were then washed once in400 μl 100% ethanol and resuspended in 45 μl of 100% ethanol. TheDNA/particle suspension was sonicated three times for one second each. 5μl of the DNA-coated gold particles was then loaded on each macrocarrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5 to 10 plates of tissue were bombarded. Membrane rupture pressure wasset at 1100 psi and the chamber was evacuated to a vacuum of 28 inchesmercury. The tissue was placed approximately 3.5 inches away from theretaining screen and bombarded once. Following bombardment, the tissuewas divided in half and placed back into liquid media and cultured asdescribed above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media containing 30 μg/ml hygromycin as selection agent. Thisselective media was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each clonallypropagated culture was treated as an independent transformation eventand subcultured in the same liquid MS media supplemented with 2,4-D (10mg/ml) and 30 μg/ml hygromycin selection agent to increase mass. Theembryogenic suspension cultures were then transferred to solid agar MSmedia plates without 2,4-D supplement to allow somatic embryos todevelop. A sample of each event was collected at this stage for PCR andquantitative PCR analysis.

Cotyledon stage somatic embryos were dried-down (by transferring theminto an empty small Petri dish that was seated on top of a 10 cm Petridish to allow slow dry down) to mimic the last stages of soybean seeddevelopment. Dried-down embryos were placed on germination solid media,and transgenic soybean plantlets were regenerated. The transgenic plantswere then transferred to soil and maintained in growth chambers for seedproduction.

Eighty two putative transgenic events were produced from transformationexperiments with the target DNA fragment QC288A. Somatic embryo samplesof the events were analyzed by quantitative PCR (qPCR), regular PCR, andSouthern to identify events with a single complete copy of thetransgene. Since DNA could be fragmented during biolistic bombardment,three major components, scp1, hpt, and yfp of QC288A were checked byqPCR. Endogenous controls were used to normalize different samples and acalibrator containing single copy of the transgene component wasincluded for calculating the relative transgene copy numbers of thesamples by comparing their relative quantifications to that of thecalibrator. Since the relative quantification values containedfractions, copy numbers were considered to be 0, 1, or 2 for values of<0.3, 0.4-1.4, or 1.5-2.4, respectively. Approximately 50% of the 82events contained one copy of the QC288A transgene based on the qPCRanalysis.

Genomic DNA samples of transgenic events were analyzed by qPCR usingTaqman technology and the universal Taqman DNA polymerase reactionmixture in a 7500 real time PCR system (Applied Biosystems, Foster City,Calif.). Relative quantification methodology was applied in single tubeduplex PCR reactions, one for the target gene and the other for anendogenous control gene to normalize the reactions across samples. After2 minutes incubation at 50° C. to activate the Taq DNA polymerase and 10minutes incubation at 95° C. to denature the DNA templates, 40 cycles of15 seconds at 95° C. and 1 minute at 60° C. were performed. A soybeanheat shock protein (hsp) gene was used as the endogenous control. Atransgenic DNA sample known containing single copy of the transgenecomponent was included as the calibrator. Three components includingscp1 promoter, hpt, yfp of the target QC288A were analyzed. Primers usedwere SEQ ID NO:8, SEQ ID NO:9, and VIC labeled MGB probe SEQ ID NO:10(Applied Biosystems) for the hsp control, SEQ ID NO:11, SEQ ID NO:12,and FAM labeled BHQ1 probe SEQ ID NO:13 (Sigma Genosis) for scp1, SEQ IDNO:14, SEQ ID NO:15, and FAM labeled BHQ1 probe SEQ ID NO:16 for hpt,SEQ ID NO:17, SEQ ID NO:18, and FAM labeled BHQ1 probe SEQ ID NO:19 foryfp.

The intactness of QC288A transgene ends was checked by regular PCR.Soybean genomic DNA was prepared from leaf discs or somatic embryosusing an extraction buffer containing 7 M urea, 1.5 M NaCl, 50 mM Tris,pH 8.0, 20 mM EDTA, and 1% N-lauroyl-sarcosine followed byphenol/chloroform extractions and isopropanol precipitations. A typical25 μl PCR reaction consisted of 10 ng genomic DNA, 200 nM of eachprimer, 200 μM dNTPs, 1×PCR buffer, and 2.5 units of High Fidelity TaqDNA polymerase (Invitrogen). A typical PCR was done at 94° C. for 3 minfollowed by 40 cycles at 94° C. for 0.5 min denaturizing, 60° C. for 1min annealing, 68° C. for 1˜3 min extension (depending on the size ofPCR amplicon), and then a final 5 min extension at 68° C. using aGeneAmp 9700 PCR system (Applied Biosystems). The 5′ end intactness ofthe QC288A transgene in target plants was analyzed with primers SEQ IDNO:23 and SEQ ID NO:24 to amplify a 657 bp band. The 3′ end intactnessof the QC288 transgene was analyzed with primers SEQ ID NO:25 and SEQ IDNO:26 to amplify a 441 bp band. Only events positive for both the PCRanalyses were selected.

Selected events were further analyzed by Southern with two probes hptand yfp. Soybean genomic DNA was digested with EcoRV, resolved in 0.7%agarose gel, and blotted to a nylon membrane using a TurboBlotter(Schleicher & Schuell Bioscience, Germany) with 20×SSC (Invitrogen) andcrosslinked by UV light. Digoxigenin labeled DNA probes were made by PCRfrom plasmid DNA templates using the PCR DIG probe synthesis kit (RocheApplied Science, Indianapolis, Ind., USA). The 794 bp hpt probe was madewith primers SEQ ID NO:28 and SEQ ID NO:29. The 693 bp yfp probe wasmade with primers SEQ ID NO:30 and SEQ ID NO:31. Southern blots werehybridized in DIG EasyHyb solution and detected with CDP-Star accordingto the manufacturer (Roche Applied Science). Hybridization signals werecaptured on BioMax light films (Eastman Kodak, New Haven, Conn., USA).

The restriction enzyme EcoRV cuts QC288A twice in the middle atpositions 2078 and 3246. To each copy of the transgene, the hpt probewould hybridize to a 2078 or larger band, and the yfp probe wouldhybridize to a 1299 bp or larger band. The Southern analysis confirmedthe copy numbers determined by qPCR for most events. Four events, eachdetermined to contain a single intact copy of QC288A by qPCR, PCR, andSouthern analyses, were selected for RMCE retransformation (Table 1).

Table 1 presents a summary of the analysis of single copy transgenicevents selected at somatic embryo stage for RMCE retransformation. Froma total of 82 events, four were selected as being single intact copyevents as determined by the qPCR, Southern, and transgene end-specificPCR. Fraction values were produced by qPCR for transgene copy numbers. Avalue less than 0.3 was considered as zero copy and a value between 0.4and 1.4 was considered as one copy. Due to the variations, differentcomponents of the same transgenic DNA were checked in order to make acopy number call. The intactness of FRT1 site was checked by PCR withprimers Scp1-S/Hygro-A (SEQ ID NO:23/SEQ ID NO:24). The intactness ofFRT87 site was checked with primers Yfp-3/Frt87-A (SEQ ID NO:25/SEQ IDNO:26).

TABLE 1 Analysis of Target Events Target Quantitative PCR PCRSouthern-EcoRV Event scp1 hpt yfp FRT1 FRT87 yfp hpt M 1.0 0.6 1.0 + + 11 A 1.1 0.6 1.1 + + 1 1 B 0.9 0.6 1.0 + + 1 1 N 1.0 0.8 1.0 + + 1 1

Example 3 Retransformation Event Creation and Characterization by PCR

Four transgenic events containing a single complete copy of the targetQC288A DNA were maintained as suspension cultures and retransformed withthe donor construct QC329 and the FLP construct QC292 at 10:1 ratiofollowing the same biolistic bombardment transformation protocoldescribed in EXAMPLE 2 except that retransformation events were selectedusing 90 ng/ml chlorsulfuron (DuPont, Wilmington, Del., USA). RMCE wouldonly occur in cells containing all three DNA QC288A, QC329, and QC292and would bring the promoter-less als coding region of QC329 downstreamof the scp1 promoter of QC288A previously placed in soybean genome forexpression and thus chlorsulfuron resistance.

The somatic embryo samples of putative retransformation events werescreened by PCR as described in EXAMPLE 2 using construct-specificprimers as depicted in FIGS. 1A-1E. Plasmid DNA of constructs QC288,QC329, QC292 were included as positive controls. An unrelated constructQC165 was used as a positive control for RMCE DNA QC288A329 since theyboth contain the same scp1:als cassette (FIG. 1C, FIG. 1E). Wild-typeDNA and no DNA template negative controls were also included (FIG. 4).

QC288A-specific PCR was done using primers SEQ ID NO:11 and SEQ ID NO:24to give a 416 bp band (FIG. 1A). Five events M-1, A-1, B-1, B-2, and N-1were positive and two events M-2 and M-3 were negative (FIG. 4A). Theresults suggested that the two negative events no longer contained thehpt component of QC288A. Event B-1 produced a much weakerQC288A-specific PCR band suggesting that it contained less QC288A DNAthan other positive events. QC288A329-specific PCR was done usingprimers SEQ ID NO:11 and SEQ ID NO:27 to give a 497 bp band. The sameprimers would give a 426 bp band to the RMCE positive control constructQC165 (FIG. 1E). The same two events M-2 and M-3, while negative forQC288A, were positive for QC288A329 suggesting that they were completeRMCE events. The weak QC288A positive event B-1 was also positive forQC288A329, suggesting that this event was a chimeric RMCE event thatstill contained some original target cells. The other four QC288Apositive events M-1, A-1, B-2, and N-1 were negative for QC288A329,suggesting that they were the original target events that had escapedthe chlorsulfuron selection as false retransformation events. Asexpected, a slightly smaller band was detected in the positive controlQC165 (FIG. 4B). QC329-specific PCR was done with primers SEQ ID NO:36and SEQ ID NO:27 to give a 1027 bp band. The same primers would give a982 bp band to the RMCE positive control construct QC165 (FIG. 1B, FIG.1E). A weak QC329-specific PCR band was detected in event M-1,suggesting that this event was likely a chimeric random integrationevent with a portion of cells harboring the QC329 DNA (FIG. 4C).QC292-specific PCR was done with primers SEQ ID NO:11 and SEQ ID NO:37to give a 368 bp band (FIG. 1D). A weak band was detected in event M-3,suggesting that this RMCE event might contain cells harboring the QC292DNA (FIG. 4D). Overall, three putative RMCE events M-2, M-3, and B-1were identified.

The three putative RMCE events M-2, M-3, and B-1 as well as their targetparents M and B were analyzed by another PCR using primers SEQ ID NO:23and SEQ ID NO:26 which would amplify a 5982 bp band from QC288A329 and asmaller 4393 bp band from QC288A (FIG. 1A, FIG. 10). As expected, anapproximately 5982 bp band was detected in the three putative RMCEevents M-2, M-3, and B-1; and an approximately 4393 bp band was detectedin their target parents M and B (FIG. 4E). More analyses as describedbelow later confirmed that only M-2 and B-1 were true RMCE events whileM-3 was indeed a SSI event with the donor DNA QC329 simply integrated atthe FRT1 site of the target DNA QC288A.

Example 4 Retransformed T0 Plant Characterization by PCR and qPCR

T0 plants were regenerated from events M-1, M-2, M-3, B-1, and B-2 andanalyzed by PCR and qPCR as described in EXAMPLE 2. DNA recombination atthe FRT1 site was checked by regular PCR with two sets of primers SEQ IDNO:11/SEQ ID NO:24 and SEQ ID NO:11/SEQ ID NO:27. The original QC288Awould be positive for SEQ ID NO:11/SEQ ID NO:24 and negative for SEQ IDNO:11/SEQ ID NO:27 while the recombination DNA QC288A329 would benegative for SEQ ID NO:11/SEQ ID NO:24 and positive for SEQ ID NO:11/SEQID NO:27 (FIG. 1A, FIG. 1C).

DNA recombination was further evaluated by the presence or absence ofQC288A and QC329 components checked by qPCR. Since the scp1 promoter isoutside of the FRT1 and FRT87 and thus not directly affected by DNArecombination, all events should be positive for scp1 qPCR. If an eventonly contained QC288A, the event would be positive for theQC288A-specific hpt, yfp qPCR. If an event contained both QC288A andQC329 in the cases of random integration and SSI, the event would bepositive for all the hpt, yfp, and cfp qPCR. A RMCE event, in which thesegment between the FRT1 and FRT87 sites of QC288A was replaced by thecorresponding segment of QC329, would be negative for theQC288A-specific hpt, yfp qPCR and positive for the QC329-specific cfpqPCR (FIG. 1A, FIG. 1B, FIG. 1C). Genomic DNA samples of theretransformation events were analyzed by qPCR for hpt, yfp as describedin EXAMPLE 2. The qPCR for cfp was done similarly using primers SEQ IDNO:20, SEQ ID NO:21, and FAM labeled MGB probe SEQ ID NO:22.

Table 2 presents the results of both the regular PCR and qPCR analysesdescribed above for T0 plants from the five retransformed lines M-1,M-2, M-3, B-1, and B-2 and for their target parent plants M and B ascontrols. The QC288A-specific PCR was done with primers 35S-277F (SEQ IDNO:11) and Hygro-A (SEQ ID NO:24) as a target DNA control. TheQC28A329-specific PCR was done with primers 35S-277F (SEQ ID NO:11) andAls-3 (SEQ ID NO:27) to check for DNA recombination at the FRT1 site.The event identities were determined by comparing all the results topredictions based on the construct maps presented in FIGS. 1A-1E. Atarget parent event would be positive for 35S-277F/Hygro-A (SEQ IDNO:11/SEQ ID NO:24) and negative for 35S-277F/Als-3 (SEQ ID NO:11/SEQ IDNO:27) PCR, positive for scp1, hpt, yfp and negative for cfp qPCR. Afalse retransformation event would be identical to its target parentsince it was the original target that had escaped retransformationselection. A random integration event would be positive for35S-277F/Hygro-A (SEQ ID NO:11/SEQ ID NO:24) and negative for35S-277F/Als-3 (SEQ ID NO:11/SEQ ID NO:27) PCR, and positive for allscp1, hpt, yfp, and cfp qPCR. An RMCE event would be negative for35S-277F/Hygro-A (SEQ ID NO:11/SEQ ID NO:24) and positive for35S-277F/Als-3 (SEQ ID NO:11/SEQ ID NO:27) PCR, and negative for hpt andyfp qPCR, and positive for scp1, and cfp qPCR. A SSI event would benegative for 35S-277F/Hygro-A and positive for 35S-277F/Als-3 PCR, andpositive for all scp1, hpt, yfp, and cfp qPCR.

TABLE 2 Analysis of T0 Plants from Retransformed Events PCR (SEQ ID NOs)Transgenic SEQ: SEQ: Quantitative PCR Event Event 11/24 11/27 scp1 hptyfp cfp Identity M + − 0.6 1.0 0.7 0.0 Target M-1 + − 0.6 0.9 0.7 1.1Random M-2 − + 0.5 0.0 0.0 1.0 RMCE M-3 − + 0.5 0.7 0.7 0.5 SSI B + −1.1 1.6 1.1 0.0 Target B-1 − + 0.6 0.1 0.3 0.6 RMCE B-2 + − 0.6 1.2 0.90.0 False In summary: M-2 and B-1 are two RMCE events derived from twoindependent target lines; M-1 is a random integration event; M-3 is aSSI event integrated at the FRT1 site; and B-2 is a falseretransformation event.

Example 5 Southern Analysis of Retransformed T0 Plants

T0 plants from retransformed lines M-1, M-2, M-3, B-1, B-2 were analyzedby Southern side-by-side with their corresponding target parents M and BT1 plants with probes yfp and cfp. Soybean genomic DNA was digested withNdeI, resolved in 0.7% agarose gel, and blotted to a nylon membraneusing a TurboBlotter™ (Schleicher & Schuell Bioscience, Germany) with20×SSC (Invitrogen) and crosslinked by UV light. Digoxigenin-labeled DNAprobes were made by PCR from plasmid DNA templates using the PCR DIGprobe synthesis kit (Roche Applied Science, Indianapolis, Ind., USA).The 693 bp yfp probe was made with primers SEQ ID NO:30 and SEQ IDNO:31. The 546 bp cfp probe was made with primers SEQ ID NO:32 and SEQID NO:33. Southern blots were hybridized in DIG EasyHyb™ solution anddetected with CDP-Star® according to the manufacturer (Roche AppliedScience). Hybridization signals were captured on Kodak™ BioMax® lightfilms (Eastman Kodak, New Haven, Conn., USA).

The restriction enzyme NdeI cuts the 4544 bp QC288A DNA once at position1119 into a 1188 bp 5′ half and a 3356 bp 3′ half, and also cuts the8533 bp QC288A329 DNA once at position 4395 into a 4394 bp 5′ half and a1739 bp 3′ half (FIG. 1A, FIG. 1C). In addition to thetransgene-specific NdeI site, the enzyme has to cut another nearby NdeIsite in soybean genomic DNA in order to produce a Southern band ofcertain size to be hybridized by a transgene probe.

The yfp and cfp probes were used to analyze the 3′ half of the transgenelocus. A target event would be positive for yfp and negative for cfp, aRMCE event would be negative for yfp and positive for cfp, and a randomintegration event or a SSI event would be positive for both yfp and cfp.More accurately, the cfp band in a RMCE sample would be 1617 bp smallerthan the corresponding yfp band in its target parent sample because the1739 bp 3′ half of QC288A329 is 1617 bp shorter than the 3356 bp 3′ halfof QC288A. As expected, the yfp probe detected single band in the targetevents M, B, the random integration event M-1, the SSI event M-3, andthe false event B-2 but not in the two RMCE events M-2 and B-1 (FIG.5A). The cfp probe detected single band in the random integration eventM-1, the SSI event M-3, and the RMCE events B-1, but three bands inanother RMCE event M-2 (FIG. 5B). As expected, the middle cfp band inM-2 is approximately 1617 bp smaller than the yfp band in M while thecfp band in B-1 is approximately 1617 bp smaller than the yfp band in B.The two extra cfp bands in M-2 are of random sizes and are likelyrandomly integrated partial copies of the cfp gene that could not bedetected by the qPCR (Table 2) that, with a 69 bp PCR amplicon, woulddetect only a small part of what the 546 bp cfp probe could detect inSouthern.

Example 6 Confirmation of DNA Recombination by Sequencing

To check if DNA recombination at FRT1 and FRT87 sites was accurate, thetransgenic gene QC288A329 was cloned by PCR amplification from M-2, M-3,and B-1. The 5′ half was amplified as a 2730 bp PCR fragment usingprimers SEQ ID NO:23 and SEQ ID NO:34 while the 3′ half was amplified asa 3351 bp PCR fragment using primers SEQ ID NO:35 and SEQ ID NO:26 (FIG.1C). A 99 bp segment between the SEQ ID NO:35 and SEQ ID NO:34 primersoverlaps the two fragments so that the entire transgene can besequenced. The PCR fragments were cloned into pCR2.1-TOPO vector with TAcloning kit according to the manufacturer (Invitrogen). Plasmid DNA wasprepared with Qiaprep plasmid DNA kit (Qiagen) and sequenced usingApplied Biosystems 3700 capillary DNA analyzer and dye terminator cycleDNA sequencing kit. Sequence assembly and alignment were done usingVector NTI suite programs (Invitrogen). Sequence searches were doneremotely using the NCBI advanced BLAST algorithm.

Since QC288A and QC288A329 sequences diverge downstream of the FRT1site, with hpt in QC288A and als in QC288A329, and upstream of the nosterminator, with yfp in QC288A and cfp in QC288A329 (FIG. 1A, C),alignment of the transgene sequences with the predicted QC288A andQC288A329 map sequences would confirm RMCE recombination at the sequencelevel. However, since the same 3351 bp SEQ ID NO:35/SEQ ID NO:26 PCRband could be obtained entirely from the donor construct QC329, the PCRusing SEQ ID NO:35 and SEQ ID NO:26 primers would not distinguish theFRT1 site SSI event M-3 from the RMCE events M-2 and B-1.

Both the predicted 5′ half 2730 bp band and 3′ half 3551 bp band weresuccessfully amplified from the three events M-2, M-3, and B-1, andsubsequently cloned and sequenced. Their sequences were identical to thepredicted QC288A329 map sequence and thus confirmed that DNArecombination occurred at the FRT1 site was accurate for the two RMCEevents M-2 and B-1 and also for the SSI event M-3.

Example 7 Analysis of T0 and T1 Plants from Selected Target Lines

Transgenic target events were produced from transformation experimentswith the target DNA fragment QC288A as described in EXAMPLE 2. Fourtarget events selected at tissue culture stage were retransformed andRMCE retransformation events were obtained as described in EXAMPLES 3-6.Simutaneously, seventy-nine T0 transgenic target plants were producedfrom thirty-three target events by regenerating 1-3 plants per event.Leaf samples of all the T0 plants were analyzed by the same qPCR, PCR,and Southern analyses described above for copy number and geneintactness confirmation. Twenty single copy events (or lines) wereselected based on the analyses and seed sets. Sixteen seeds from one T0plant from each of ten lines selected from the twenty were planted toget T1 plants. Leaf samples of all the T1 plants were analyzed by threeqPCR analyses specific to the scp1 promoter, ubq10 promoter, and yfpgene to check for segregation of the QC288A transgene. Homozygous T1plants were obtained from eight lines. Three homozygous target lines A,B, and C were selected for RMCE retransformation experiments.

Table 3 presents the results of qPCR, PCR, and Southern analyses on T0plants from three transgenic target lines. Fraction values were producedby qPCR for transgene copy numbers. A value less than 0.3 was consideredas zero copy, a value between 0.4 and 1.3 was considered as one copy,and a value between 1.4 and 2.3 was considered as two copies. Multiplecomponents of the target DNA (FIG. 1A) were checked in order to make avalid copy number call. The intactness of FRT1 site was checked by PCRwith primers Scp1-S/Hygro-A (SEQ ID NO:23/SEQ ID NO:24) to give a 657 bpband. The intactness of FRT87 site was checked with primersYfp-3/Frt87-A (SEQ ID NO:25/SEQ ID NO:26) to give a 1441 bp band. Thefull length transgene was checked with primers Scp1-S/Frt87-A (SEQ IDNO:23/SEQ ID NO:26) to give a 4393 bp band (FIG. 1A). All three linescarry a complete single copy of the transgenic target DNA.

TABLE 3 Analysis of T0 Plants from Target Lines Target Quantitative PCTPCR Southern Line scp1 hpt yfp FRT1 FRT87 Full hpt yfp A 1.1 0.61.1 + + + 1 1 B 0.9 0.6 1.0 + + + 1 1 C 1.0 1.1 0.8 + + + 1 1

Table 4 presents the results of qPCR analyses on homozygous T1 plantsfrom the three transgenic target lines. As above, a value less than 0.3was considered as zero copy, a value between 0.4 and 1.3 was consideredas one copy representing hemizygous plants, and a value between 1.4 and2.3 was considered as two copies representing homozygous plants.

TABLE 4 Analysis of Homozygous T1 Plants from Target Lines TargetQuantitative PCT Line scp1 ubq10 yfp A 1.6 1.8 1.5 B 1.6 2.0 1.6 C 1.82.0 1.5

Example 8 Target Line Border Sequencing

Genomic DNA fragments bordering the QC288A transgene on both the 5′ endand 3′ end of six target lines were obtained by PCR amplification andcloned for sequencing.

The GenomeWalker kit (ClonTech, Mountain View, Calif., USA) was used toacquire the genomic DNA sequences bordering the transgenic genes. DNAsamples of each target line were digested separately with blunt endrestriction enzymes EcoRV, DraI, HpaI, and StuI before adding theGenomeWalker™ DNA adaptors. The first round of PCR was done with theadaptor-specific primer AP1 (SEQ ID NO:65) provided in the kit andQC288A-specific primers, Scp1-A (SEQ ID NO:66) for the 5′ end border andVec-S1 (SEQ ID NO:67) for the 3′ border, respectively. The second roundof PCR was done with the adaptor-specific primer AP2 (SEQ ID NO:68)provided in the kit and QC288A-specific primers, Scp1-A4 (SEQ ID NO:69)for the 5′ end border and Vec-S2 (SEQ ID NO:70) for the 3′ border,respectively. Specific DNA fragments amplified by the second round PCRwere cloned into pCR2.1-TOPO® vector with TA cloning kit according tothe manufacturer (Invitrogen). Plasmid DNA was prepared with Qiaprep®plasmid DNA kit (Qiagen) and sequenced using Applied Biosystems 3700capillary DNA analyzer and dye terminator cycle DNA sequencing kit.Sequence assembly and alignment were done using VECTOR NTI® suiteprograms (Invitrogen). Sequence searches were done remotely using theNCBI advanced BLAST algorithm.

The bordering genomic DNA sequences and truncations of the transgeneends were revealed by aligning the PCR clone sequences to QC288A mapsequence. Various lengths of bordering genomic DNA sequences wereobtained and the truncations of transgene ends were revealed to be minorfor all the six target lines. Target lines A, B, C, and N lost 5, 17,22, and 2 bp of the 5′ end of the transgene, and 0, 49, 11, and 0 bp ofthe 3′ end of the transgene, respectively. Genomic DNA sequences of 601bp (SEQ ID NO:55), 984 bp (SEQ ID NO:57), 496 bp (SEQ ID NO:61), and 452bp (SEQ ID NO:59) bordering the 5′ end of the transgene, and 2588 bp(SEQ ID NO:56), 1305 bp (SEQ ID NO:58), 543 bp (SEQ ID NO:62), and 377bp (SEQ ID NO:60) bordering the 3′ end of the transgene were obtainedfor the three target lines A, B, C and N, respectively. The borderinggenomic DNA sequences were used to search NCBI nucleotide collection(nr/nt) database by BLASTN to determine if any endogenous gene ofimportance was interrupted by the transgene insertion. No significanthomology to any known gene was found for any of the three target lines.The bordering genomic DNA sequences were also used to design primers forborder-specific PCR analysis of the target lines and RMCE events derivedfrom the target lines in subsequent retransformation.

Example 9 RMCE Event Creation Using Suspension Cultures Derived fromHomozygous T1 Target Plants

Suspension cultures were initiated from developing embryos fromhomozygous T1 plants of the three target lines A, B, and C andretransformed by co-bombardments with the donor construct QC329 and FLPexpression construct QC292 plasmid DNA.

The homozygous transgenic target line cultures were retransformed withthe donor construct QC329 and the Flp construct QC292 at a 10:1 ratiofollowing the biolistic bombardment transformation protocol except using90 ng/ml chlorsulfuron (DuPont, Wilmington, Del., USA) as the selectionagent. RMCE could only occur in cells containing all three DNAs, QC288A,QC329, and QC292, and would bring the promoter-less als coding region ofQC329 downstream of the scp1 promoter of QC288A previously placed insoybean genome through DNA recombination for expression and thuschlorsulfuron resistance.

Putative retransformation events were selected by chlorsulfuronresistance and checked for reporter gene cfp expression under afluorescent microscope. CFP positive events were sampled at somaticembryo stage and screened by a common PCR with primers 35S-277F (SEQ IDNO:11) and Als-3 (SEQ ID NO:27) to amplify a RMCE-specific 497 bp bandto check for DNA recombination around the FRT1 site. Then the eventswere analyzed by construct-specific qPCR to confirm DNA recombination atFRT1 site and to check for the presence of target, donor, and Flp DNA.RMCE, target, and donor-specific qPCR assays were designed around theFRT1 recombination site in each DNA construct. RMCE-specific qPCRemployed 288A-1F (SEQ ID NO:71), Als-163R (SEQ ID NO:72) primers andFAM-labeled BHQ1 probe Als-110T (SEQ ID NO:73). Target-specific qPCRemployed 288A-1F (SEQ ID NO:71), Hygro-116R (SEQ ID NO:74) primers andFAM-labeled BHQ1 probe Hygro-79T (SEQ ID NO:75). Donor-specific qPCRemployed 329-1F (SEQ ID NO:76), Als-163R (SEQ ID NO:72) primers andFAM-labeled BHQ1 probe Als-110T (SEQ ID NO:73). Another qPCR assayspecific to the Flp construct QC292 employed Ucp3-57F (SEQ ID NO:77),Flp-A (SEQ ID NO:78) primers and FAM-labeled BHQ1 probe OMEGA5UTR-87T(SEQ ID NO:79).

Border-specific PCR analyses specific to each target line 5′ end and 3′end borders were done on corresponding events to check DNA recombinationat FRT1 site, at FRT87 site, and also between FRT1 and FRT87 sites. TheRMCE 5′ border-specific PCR employed the common antisense primer Als3(SEQ ID NO:27) and a target line 5′ border sequence-specific senseprimer, 53-1S1 (SEQ ID NO:80) for target line A, 70-1S (SEQ ID NO:81)for target line B, and 8H-ScaS1 (SEQ ID NO:82) for target line C. TheRMCE 3′ border-specific PCR employed the common sense primer Cyan-1 (SEQID NO:83) and a target line 3′ border sequence-specific antisenseprimer, 53-1A (SEQ ID NO:84) for target line A, 70-1A (SEQ ID NO:85) fortarget line B, and 8H-VecA (SEQ ID NO:86) for target line C. The target5′ border-specific PCR employed the same target line 5′ bordersequence-specific sense primers but a target cassette-specific commonantisense primer Hygro-A (SEQ ID NO:24). The target 3′ border-specificPCR employed the same target line 3′ border sequence-specific antisenseprimers but a target cassette-specific common sense primer Yfp-3 (SEQ IDNO:25). The full length PCR employed the same target line 5′ bordersequence-specific sense primer and the same target line 3′ bordersequence-specific antisense primer for each of the three target lines tosimultaneously amplify a small excision band, the full target transgeneband, and the full RMCE transgene band. Expected sizes of the RMCE 5′end-specific, RMCE 3′ end-specific, Target 5′ end-specific, Target 3′end-specific, full length Excision, full length Target, and full lengthRMCE PCR are 1117, 1351, 1036, 732, 1307, 5063, and 6652 bp for targetline A events; 967, 1180, 886, 561, 986, 4742, and 6331 bp for targetline B events; and 1018, 1294, 937, 675, 1151, 4907, and 6496 bp fortarget line C events.

FIG. 6 presents the results of analyses of three retransformation eventsderived from target line A, including a CFP negative event A3 as anegative control, four retransformation events from target line B, andthree retransformation events from target line C. For example, event A1was positive for CFP expression and positive for DNA recombination atFRT1 site as determined by the common PCR. For construct-specific qPCRanalyses, event A1 was positive for RMCE, contained one copy of donorDNA, and was free of either target or Flp DNA. For border-specific PCRanalyses, event A1 was positive for both the 5′ end and 3′ end assaysspecific to RMCE, and negative for both the 5′ end and 3′ end assaysspecific to the target. Full length PCR from the 5′ end border to the 3′end border amplified a small band specific to excision and failed toamplify any band specific to the target or RMCE. The exicision was theoutcome of DNA recombination between the FRT1 and FRT87 sites of eitherthe target DNA QC288A, RMCE DNA QC288A329, or the intermediates SSI DNAQC288A329FRT1 and QC288A329FRT87 with all components flanked by theborder FRT1 and FRT87 sites excised (FIG. 3D). The restored FRT sitecould be either FRT1 or FRT87 depending on the DNA strands crossing overposition. Based on the above analyses, event A1 was a RMCE/Excisionevent contaminated with one randomly integrated copy of the donor DNA.Target DNA on two homologous chromosomes of the homozygous target linewas replaced by RMCE on one chromosome and by excision on the othersince target DNA was no longer detectible by either target-specific qPCRor target 5′ border, 3′ border, and full length border-specific PCRanalyses.

Following similar analyses described above for event A1, the followingconclusions were made for the other nine events listed in FIG. 6. EventA2 was a RMCE/Excision containing a randomly integrated copy of donorand a randomly integrated copy of Flp DNA. Event A3 was a homozygousTarget escape carrying about five randomly integrated copies of thedonor DNA. Events B1, B2, and B4 were all RMCE/Excision events with norandomly integrated copies of donor or Flp DNA. Event B3 was aRMCE/Excision containing randomly integrated donor DNA. Event C1 was anincomplete RMCE/Excision still containing the target DNA as detected bythe 5′ end and 3′ end border-specific PCR analyses. The target-specificqPCR detected only 0.01 copy of target DNA for event C1. Events C2 andC3 were RMCE/RMCE (homozygous RMCE) with both the targets on homologouschromosomes being converted to RMCE since the RMCE-specific qPCRdetected two copies while the target-specific qPCR as well as threeborder-specific PCR analyses failed to detect either any target-specificor excision-specific band. Both C2 and C3 events also contained randomlyintegrated donor DNA.

As examples, border-specific PCR analyses on events A1, A2, and A3 areshown in FIGS. 7A-7E. Genomic DNA samples extracted from somatic embryosof the three events were analyzed by RMCE-specific PCR specific to the5′ end border (FIG. 7A), and specific to the 3′ end border (FIG. 7B).Events A1 and A2 were positive for the 1117 bp RMCE 5′ endborder-specific band and also positive for the 1351 bp RMCE 3′ endborder-specific band. Event A3 was negative for either the 5′ end borderor 3′ end border band (FIG. 7A, FIG. 7B). When the same DNA samples wereanalyzed by Target-specific PCR specific to the 5′ end border (FIG. 7C),and specific to the 3′ end border (FIG. 7D), events A1 and A2 failed toproduce any band. In contrast, event A3 was positive for the 1036 bpTarget 5′ end border-specific band and also positive for the 732 bpTarget 3′ end border-specific band (FIG. 7C, FIG. 7D). The full length5′ end border to 3′ end border PCR amplified only the 1307 bpExcision-specific band for events A1 and A2 but not for event A3 (FIG.7E). The expected 6652 bp RMCE-specific band that should exist in eventsA1 and A2 failed to be amplified due to the dominant competitionadvantage of the small Excision-specific products in the same PCRreactions. The same full length border-specific PCR amplified the 5063bp Target-specific band from event A3 (FIG. 7E). Wild type DNA and watertemplates were included as negative controls in all the analyses.

Example 10 Characterization of T0 Plants from RMCE Events

T0 plants were regenerated from the RMCE events and their leaf sampleswere subjected to the same construct-specific qPCR and border-specificPCR analyses described in EXAMPLE 9. The analysis results of T0 plantsof three independent events are listed in FIG. 8. Three plants A2-1,A2-2, and A2-3 of event A2, four plants C2-1, C2-2, C2-3, and C2-4 ofevent C2, and two plants C3-1, and C3-2 of event C3 all retained thesame molecular signatures of their respective events A2, C2, and C3 thatwere revealed at the somatic embryo stage (FIG. 6). The differences ofqPCR copy number values between the somatic embryo samples and the T0plant samples were in the normal range of experimental variations.

Border-specific PCR analyses on plants listed in FIG. 8 are shown inFIGS. 9A-9E. Results of the RMCE-specific PCR specific to the 5′ endborder (FIG. 3A), specific to the 3′ end border (FIG. 9B), theTarget-specific PCR specific to the 5′ end border (FIG. 9C), specific tothe 3′ end border (FIG. 9D), and the full length 5′ end border to 3′ endborder PCR (FIG. 9E) for the T0 plant samples A2-1, A2-2, A2-3, A2-4,C2-1, C2-2, C2-3, C3-1, and C3-2 all matched the somatic embryo DNA A2,and C2 RMCE positive controls as well as the previous border-specificPCR analyses results (FIG. 6). Since A2 and its T0 plants A2-1, A2-2,A2-3, and A2-4 are all heterozygous RMCE/Excision, the full length PCRonly amplified the small Excision-specific band but not the expected6652 bp large RMCE band, since PCR amplification favored the small band.In contrast, the full length PCR amplified the 6496 bp RMCE band fromhomozygous RMCE events C2, C3 (not shown in FIGS. 9A-9E) and their T0plants C2-1, C2-2, C2-3, C3-1, and C3-2 in the absence of the otherwiseexpected 1151 bp Excision-band. The Target parent DNA samples A and Cwere included as positive controls for Target and negative controls forRMCE. Wild-type DNA (wt) and no DNA template (H₂O) were included asnegative controls.

Since the Target QC288A and the RMCE QC288A329 sequences divergedownstream of the FRT1 site, with hpt in QC288A and als in QC288A329,and upstream of the nos terminator, with yfp in QC288A and cfp inQC288A329, alignment of the transgene sequences with the predictedQC288A and QC288A329 map sequences should confirm RMCE recombination atthe sequence level. The predicted sequences surrounding the FRT1 sitewere aligned to show the differences among Target, RMCE, and Excisiondownstream of the FRT1 site (FIG. 10A). Depending on the crossing-overposition, Excision resulted from the recombination between FRT1 andFRT87 sites could restore either the FRT87 site or the FRT1 site (Groth,A. C. and Calos, M. P., J. Mol. Biol. 335:667-678 (2003)). The predictedsequences surrounding the FRT87 site were aligned to show thedifferences between Target and RMCE upstream of the nos terminator onthe 5′ end of the FRT87 site (FIG. 10B). The sequences of the genomicDNA part upstream of the scp1 promoter on the 5′ end or downstream ofthe QC288A 3′ end, though not shown, were different between Target lineA and line C and were included in the alignment analyses describedbelow.

The 21 DNA fragments amplified from seven representative samples Targetparents A, C, RMCE events A2, C2 somatic embryos, and RMCE T0 plantsA2-1, C2-1, and C3-1 by the five border-specific PCR analyses werecloned and up to 4 clones derived from each fragment were sequenced torule out sequence mutations caused by PCR (FIGS. 9A-9E). The transgenicgene sequences were aligned with predicted sequences of Target, RMCE,and Excision to confirm accurate DNA recombination around the FRT1 andFRT87 sites. Sequences obtained from the border-specific PCR DNAfragments were identical to their predicted corresponding sequences. The5′ end border-specific PCR fragments sequences of RMCE samples A2-1, A2,C2-1, C3-1, and C2 (FIG. 9A) matched the RMCE sequences surrounding theFRT1 site (FIG. 10A). The 3′ end border-specific PCR fragments sequencesof RMCE samples A2-1, A2, C2-1, C3-1, and C2 (FIG. 9B) matched the RMCEsequences surrounding the FRT87 site (FIG. 10B). The 5′ endborder-specific PCR fragments sequences of target samples A and C (FIG.9C) matched the target sequences surrounding the FRT1 site (FIG. 10A).The 3′ end border-specific PCR fragments sequences of target samples Aand C (FIG. 9D) matched the target sequences surrounding the FRT87 site(FIG. 10B). The excision-specific PCR fragments sequences of A2-1, andA2 (FIG. 9E) matched one of the predicted excision-specific sequencescontaining the FRT1 site (FIG. 10A). Both the 5′ and 3′ ends of the fulllength Target fragment sequences of A and C or the full length RMCEsequences of C2-1, C3-1, and C2 (FIG. 9E) matched the ends of theoriginal QC288A sequence or the predicted QC288A329 sequence,respectively (FIGS. 10A-10B).

Example 11 Characterization of T1 Plants from RMCE Events

T1 seeds harvested from T0 plants A2-1, A2-2, A2-3, A2-4, C3-1, and C3-2were germinated and the T1 plants were analyzed by the sameconstruct-specific qPCR analyses done previously on their parents. Sincethe four T0 plants of event A2 were identical and the two T0 plants ofevent C3 were identical based on previous analyses (FIG. 8 and FIGS.9A-9E), a total of 42 T1 plants derived from the four A2 T0 plants and atotal of 48 T1 plants derived from the two C3 T0 plants were treated astwo populations for segregation analysis. Since all four A2 T0 plantswere confirmed to be heterozygous for RMCE/Excision and contaminatedwith Donor and Flp DNA (FIG. 8), the Excision should segregate away fromRMCE, and the Donor and Flp should also segregate if they were in adifferent site that was not linked to the RMCE/Excision target site. TheRMCE-specific qPCR would detect two copies, one copy, or null of RMCEfor plants that are RMCE/RMCE, RMCE/Excision, and Excision/Excision,respectively. Similarly, Target-specific qPCR, Donor-specific qPCR, andFlp-specific qPCR would detect two copies, one copy, or null of Target,Donor, or Flp for homozygous (homo), hemizygous (hemi), or null of theTarget, Donor, or Flp gene, respectively.

Of the forty-two A2 T1 plants, the RMCE/Excision site segregated astwelve RMCE/RMCE, eighteen RMCE/Excision, and twelve Excision/Excision.The Donor and Flp were apparently linked and segregated independentlyfrom the RMCE as fifteen homozygous, sixteen hemizygous, and elevennull. Seven plants were RMCE/Excision and free of any Donor or Flp. Oneplant was clean homozygous RMCE/RMCE and free of any Donor or Flp DNA.Consistent with previous analyses at T0 generation, all A2 T1 plantswere free of the Target gene. All forty-eight C3 T1 plants werehomozygous RMCE/RMCE and free of any Target or Flp consistent with theconclusion that the C3 T0 parent plants were homozygous RMCE/RMCE freeof any Target or Flp (FIG. 8). The Donor was not linked to the RMCE siteand segregated as twelve homozygous, twenty-four hemizygous, and twelvenull. So twelve C3 T1 plants were clean homozygous RMCE/RMCE and free ofany Donor or Flp DNA.

In summary, clean homozygous RMCE plants free of Donor, Target, or FlpDNA were obtained at the T1 generation from the retransformation ofmultiple Target lines by FLP/FRT recombinase mediated cassette exchange.

Example 12 Transgene Stacking Through Multiple Rounds of RMCE

With the success of RMCE confirmed, one can design strategies involvingrepeated RMCE to place multiple transgenes at the same genomic sitewhere the target DNA QC288A has inserted. Two groups of transgenes canbe stacked through two rounds of RMCE as illustrated in FIG. 11A andFIG. 11B. Three incompatible FLP recognition sites FRT1, FRT12, andFRT87 as exemplified by QC422 in FIG. 11A are incorporated in a firstdonor construct designed for retransformation of selected target QC288Atransgenic lines with chlorsulfuron selection. The group 1 of transgenicgenes can be cloned between the FRT12, and FRT87 sites. RMCE will happenbetween the target QC288A in the genome and the first donor QC422 or itsderivative through the FRT1 and FRT87 sites. The first round RMCEretransformation events will contain, in addition to group 1 transgenes,three FLP recognition sites FRT1, FRT12, and FRT87 (FIG. 12A). Selectedfirst round RMCE retransformation events will be retransformed with asecond donor construct containing two FLP recognition sites FRT1 andFRT12 as exemplified by QC429 in FIG. 11B with hygromycin selection. Thegroup 2 transgenic genes can be cloned upstream of the FRT12 site. Thesecond round of RMCE will happen between the first RMCE DNA in thegenome and the second donor QC429 or its derivative through the FRT1 andFRT12 sites. The second RMCE DNA will contain both group1 and group2transgenic genes and the three FLP recognition sites FRT1, FRT12, andFRT87 at the same genomic site (FIG. 12B). Since the three FLPrecognition sites are not compatible to each other, the transgenes arestable.

A third group of transgenic genes can be similarly stacked at the samegenomic site. The first round of RMCE and the first donor construct willbe the same as described above. The second donor construct for thesecond RMCE will also contain three FLP recognition sites as exemplifiedby QC459 in FIG. 13A with one of the three FRT sites FRT1, FRT6 andFRT12 never being used before i.e. FRT6. The group 2 of transgenic genescan be cloned between the FRT6 and FRT12 sites. Selected first roundRMCE retransformation events will be retransformed with the second donorDNA with hygromycin selection. The second RMCE will happen between thefirst RMCE DNA in the genome and the second donor DNA though the FRT1and FRT12 sites. The second round RMCE DNA will contain both group1 andgroup2 transgenic genes and four FLP recognition sites FRT1, FRT6,FRT12, and FRT87 at the same genomic site (FIG. 14A). Since the four FLPrecognition sites are not compatible to each other, the transgenes arestable.

A third RMCE retransformation will be required to stack the third groupof transgenic genes. Selected second round RMCE retransformation eventswill be retransformed with a third donor construct as exemplified byQC428 in FIG. 13B with two FLP recognition sites FRT1 and FRT6 withchlorsulfuron selection. The group 3 of transgenic genes can be clonedupstream of the FRT6 site. The third round RMCE DNA will contain allgroup 1, group 2, and group 3 transgenic genes and four FLP recognitionsites FRT1, FRT6, FRT12, and FRT87 at the same genomic site (FIG. 14B).Since the four FLP recognition sites are not compatible to each other,the transgenes are stable.

Following the same strategy described above, more groups of transgenescan be stacked at the same genomic site using more incompatible FRTsites by additional rounds of RMCE retransformation.

Example 13 Construction of Vectors for Multiple Rounds of RMCE

The FRT12 recombination site was constructed by annealing two 106 bpcomplementary oligos (SEQ ID NO:38 and SEQ ID NO:39) engineered withmultiple cloning sites (MWG-Biotech AG, Bridgeport, Calif., USA). TheXmaI/FseI FRT12 DNA fragment was cloned into the XmaI/FseI sites ofconstruct QC408 to make QC422 (SEQ ID NO:42; FIG. 11A) containingFRT1:als:pinII:FRT12-FRT87. QC429 (SEQ ID NO:43; FIG. 11B) and itsintermediates and derivatives were made via multiple steps using routinecloning techniques as described in EXAMPLE 1.

The FRT6 recombination site was made by annealing two 106 bpcomplementary oligos (SEQ ID NO:40 and SEQ ID NO:41) engineered withmultiple cloning sites (MWG-Biotech AG). The AscI/XmaI FRT6 DNA fragmentwas cloned into the AscI/XmaI sites of construct QC408 to make QC430containing FRT1:als:pinII:FRT6-FRT87. QC459 (SEQ ID NO:44; FIG. 13A),QC428 (SEQ ID NO:45; FIG. 13B) and their intermediates and derivativeswere made via multiple steps using routine cloning techniques asdescribed in EXAMPLE 1.

Example 14 Creation of Target Sites During Development of TransgenicProduct Lines

Since target lines for RMCE are created using traditional transformationmethod, which will place the target DNA in the genome randomly,significant effort is required to produce and characterize multipletarget events to identify a target line that meets desired criteria. Theprocess may take several years and require as much effort as developinga trait-containing transgenic product line. If the process of creating atarget line can be combined with the development of a trait-containingtransgenic product line, a target site will be obtained as a by-productonce the transgenic product line is selected and well characterized.Furthermore, a trait gene or a group of genes responsible for the traitis already placed at the site making it convenient to stack new traitsthrough RMCE. A common selectable marker gene cassette is usually usedfor plant transformation to facilitate the selection of transformedevents, such as the 35S:hpt and sams:als cassettes used in soybeantransformation (US patent publication WO 00/37662) and the 35S:bar andubiq:gat cassettes used in maize transformation. Consequently, twoincompatible recombinase recognition sites such as FRT1 and FRT87 can beincorporated in the selectable marker gene cassette which can then belinked to any trait gene of interest for transformation. Once integratedin a plant genome the incorporated FRT1 and FRT87 sites can be used forRMCE.

Using the promoter trap design, the soybean transformation selectablemarker gene cassette sams:als can be modified to include a FRT1 sitebetween the sams promoter and the als coding region and a FRT87 sitedownstream of the als terminator exemplified by construct QC448 (FIG.15A). Multiple restriction sites can be engineered upstream of the samspromoter and also downstream of the als terminator for the cloning oftrait genes of interest. Once placed in soybean genome thesams-FRT1:als-FRT87 cassette can be used as a target site for RMCE withany donor construct containing a promoterless marker gene such asFRT1-hpt-FRT87. DNA construct QC448 (FIG. 15A) was made from constructscontaining components such as FRT1, FRT87, sams promoter, als codingsequence etc. described in EXAMPLES 1 and 7 via multiple steps usingroutine molecular cloning techniques described in EXAMPLE 1. First,QC446 was made by cloning the 1283 bp SphI/FseI fragment containing theals term from pZSL141 into the SphI/FseI sites of QC408. Then the 2790bp HpaI/NotI FRT1:als:als term-FRT87 fragment of QC446, the HpaIdigestion was partial, was cloned into the NcoI/NotI sites of QC431 tomake QC447 containing sams-FRT1:als:als term-FRT87, the NcoI site wasfilled in by Klenow polymerase. The sams-FRT1:als:als term-FRT87cassette was released with XmaI/NotI from QC447 and moved to theXmaI/NotI sites pZSL141 to make the final plasmid QC448 from which thecassette could be conveniently released with AscI digestion for DNAfragment preparation.

The Gateway® cloning technology, which is based on the lambda phagesite-specific recombination system (Invitrogen), can be utilized to linkthe sams-FRT1:als-FRT87 marker gene to trait genes. The construct QC448(FIG. 15A) was cut at the 5′ end of the sams promoter with SmaIdigestion. A Gateway® conversion DNA fragment containing the attR1 andattR2 recombination sites (Invitrogen) was inserted to the SmaI site ofQC448 by blunt end ligation with T4 DNA ligase to make construct QC449and QC449i, with the Gateway® DNA fragment inverted, as the destinationvectors (FIG. 15B, FIG. 15C). Trait genes will need to be first clonedbetween two corresponding recombinase recognition sites attL1 and attL2as an entry vector. In vitro recombination catalyzed by LR clonasebetween the attL sites on an entry vector and the attR sites on thedestination vector will result in the linkage of the trait genes to themarker gene in tandem or diverse orientation depending on the relativeorientations of the attL and attR sites.

An improved version of QC448 was made by adding stop codons in all openreading frames on each end of the sams:als cassette to form QC477containing ORFSTOP-B-sams-FRT1:als:als term:FRT87-ORFSTOP-A (FIG. 16A).To remove a few extra base pairs between the FRT1 and the als gene, the888 bp KpnI/EcoRI fragment of pZSL91 was moved to the SpeI/EcoRI sitesof QC446 to form QC474. Both the SpeI and KpnI sites were first treatedwith mung bean nuclease to become blunt. The sams-FRT1:als:alsterm:FRT87 fragment of QC474 was released with NotI complete digestionand HpaI partial digestion and cloned into the NcoI and NotI sites ofQC431 to form QC475, the NcoI site was first blunted with mung beannuclease. ORFSOPTA-B (SEQ ID NO:88), with stop codons in all openreading frames, was synthesized as an oligo duplex with appropriatecloning sites incorporated on the ends and in the middle (MWG-BiotechAG). The duplex was digested with XhoI/SpeI and cloned into theXhoI/XbaI sites of pZSL141 to form QC476. Finally, the 4128 bp NotI/SpeIsams-FRT1:als:als term:FRT87 fragment of QC475 was moved to theNotI/SpeI sites of QC476 to form QC477 (FIG. 16A).

A Gateway® conversion DNA fragment containing the attR1 and attR2recombination sites (Invitrogen) was inserted to the PmeI sites of QC477by blunt end ligation with T4 DNA ligase to make construct QC478 andQC478i, with the Gateway® DNA fragment inverted, as the destinationvectors (FIG. 16B, FIG. 16C). Another Gateway® conversion DNA fragmentcontaining the attR3 and attR4 recombination sites (Invitrogen) wasinserted into the PmeI sites of QC477 by blunt end ligation with T4 DNAligase to make construct QC479 and QC479i, with the Gateway® DNAfragment inverted, as the destination vectors (FIG. 16D, FIG. 16E). Thedestination vectors QC479 and QC469i can accept DNA fragments previouslycloned in entry vectors containing the attL3 and attL4 sites by LRclonase catalyzed in vitro DNA recombination.

Example 15 Stacking of Fatty Acid Modifying Genes and Amino AcidModifying Genes at the Target B Site by Two Rounds of SSI

A retransformation event designated “B-5”, produced from theretransformation of the target B culture with the donor DNA QC329(EXAMPLE 3), was confirmed by multiple PCR and qPCR analyses to be aRMCE event. The B-5 event containing the QC288A329 (FIG. 1C) transgeneswas regenerated into fertile T0 plants. Homozygous T1 plants of B-5 wereidentified by qPCR and their developing embryos were used to initiatenew embryogenic cultures for gene stacking experiments using donor DNAQC436 for the first round of SSI. The QC436 construct contains apromoter-less selectable marker gene HPT between the FRT1 and FRT12sites, and between the FRT12 and FRT87 sites inverted repeats of thesoybean delta 9 desaturase gene fragment (GM-FAD2-1 (TR1)) andthioesterase gene fragment (GM-THIOESTERASE 2 (TR4)) controlled by acommon promoter KTI3 (FIG. 17A). Since the target DNA QC288A329 does notcontain a FRT12 site, RMCE between the target QC288A329 and the donorQC436 DNA can only happen between the two FRT1 sites and the two FRT87sites. Consequently, all the components between the FRT1 and FRT87 sitesof QC288A329 can be replaced by the components between the FRT1 andFRT87 sites of QC436. The third recombination site FRT12 of QC436 issimultaneously introduced into the target B locus. Multipleretransformation events were produced and confirmed to be RMCE events byPCR and qPCR analyses (similar to EXAMPLES 3 and 4). Fatty acidprofiling on somatic embryos of the QC288A436 (FIG. 17C)retransformation events revealed significantly elevated oleic acid(18:1) content, which is the phenotype expected for suppression of theendogenous delta9 desaturase and thioesterase 2 genes. One QC288A436RMCE event culture, designated “B-5-3”, was selected as the new targetfor next round SSI using the second donor DNA QC438.

The B-5-3 culture was directly retransformed with the donor DNA QC438 assimilarly described in EXAMPLE 3. QC438 contains only two recombinationsites, FRT1 and FRT12. The promoter-less selectable gene ALS and fourother complete transgenes are flanked by the same FRT1 and FRT12 sites(FIG. 17B). The expression of the Yarrowia diacylglycerolacyltransferase gene (YL-DGAT1) is useful for the conversion of fattyacids to triacylglycerol to increase overall oil content. The expressionof the other three genes, barley high lysine (BHL8), Corynebacteriumglutamicum dihydrodipicolinate synthetase gene (CORYNE DAP A), andsoybean cysteine synthase gene (GM-CGS (TR1)), are useful to increasethe content of essential amino acids such as lysine and methionine.Retransformation events were selected by their resistance tochlorsulfuron and analyzed by multiple PCR and qPCR analyses (similar toEXAMPLES 3 and 4, with more gene-specific primers). One event,designated “B-5-3-2”, was confirmed to be a RMCE stacking eventcontaining the ALS selectable marker gene and the four traits genesYL-DGAT1, BHL8, CORYNE DAP A, and GM-CGS (TR1) of the donor QC438. TheFAD2-1 and thioesterase 2 cosuppression cassette delivered by donorQC436 during the previous SSI remained intact. Fatty acid profiling ofsomatic embryo samples of B-5-3-2 detected the expected phenotypes ofhigh oleic acid and high oil contents. Western analysis of somaticembryo or T0 plant leaf samples indicated the expression of all threegenes, BHL8, CORYNE DAP A, and GM-CGS (TR1), which are designed toimprove lysine and methionine contents.

The invention claimed is:
 1. A soybean cell, plant or seed having stablyincorporated in its genome a transfer cassette genetically linked to achromosomal region comprising SEQ ID NO:80 or SEQ ID NO:84, wherein thetransfer cassette comprises at least two non-identical recombinationsites, wherein the transfer cassette further comprises a polynucleotideencoding a selectable marker protein-coding sequence bounded by a firstrecombination site and a second non-identical recombination site.
 2. Thesoybean cell, plant or seed of claim 1, where said transfer cassette isgenetically linked to a chromosomal region comprising SEQ ID NO:80 and84.
 3. The soybean cell, plant or seed of claim 1, wherein the transfercassette further comprises a third non-identical recombination sitebounded by the selectable marker protein-coding sequence and the secondnon-identical recombination site.
 4. The soybean cell, plant or seed ofclaim 3, wherein the transfer cassette further comprises at least oneexpression cassette of interest, wherein the at least one expressioncassette of interest is bounded by the third non-identical recombinationsite and the second non-identical recombination site.
 5. The soybeancell, plant or seed of claim 1, wherein said selectable markerprotein-coding sequence encodes a protein selected from the groupconsisting of a hygromycin phosphotransferase, a sulfonylurea-tolerantacetolactate synthase, and a sulfonylurea-tolerant acetolactate synthasethat has an amino acid sequence comprising SEQ ID NO:63 or SEQ ID NO:64.6. The soybean cell, plant or seed of claim 1, wherein at least one ofsaid non-identical recombination sites is selected from the groupconsisting of FRT1 (SEQ ID NO:50), FRT5 (SEQ ID NO:51), FRT6 (SEQ IDNO:52), FRT12 (SEQ ID NO:53) and FRT87 (SEQ ID NO:54).
 7. A method forstacking of multiple expression cassettes of interest into a specificchromosomal site in a soybean genome, said method comprising: a)obtaining a transgenic soybean cell comprising a target site geneticallylinked to a chromosomal region comprising SEQ ID NO:80 or SEQ ID NO:84,wherein said target site comprises a first selectable markerprotein-coding sequence, wherein the first selectable markerprotein-coding sequence is bounded by a first recombination site and asecond non-identical recombination site; b) introducing into thetransgenic soybean cell of step (a) a transfer cassette, wherein saidtransfer cassette comprises a second selectable marker protein-codingsequence, wherein the second selectable marker protein-coding sequenceis bounded by the first recombination site and the second non-identicalrecombination site, and further wherein the transfer cassette furthercomprises at least one expression cassette of interest, wherein the atleast one expression cassette of interest is bounded by the secondselectable marker protein-coding sequence and the second non-identicalrecombination site; and c) providing a recombinase that recognizes andimplements recombination at the non-identical recombination sites. 8.The method of claim 7, wherein the transfer cassette further comprises athird non-identical recombination site bounded by the second selectablemarker gene and the at least one expression cassette of interest.
 9. Themethod of claim 7 step (c), wherein providing said recombinase comprisestransiently expressing within said soybean cell an expression cassettecomprising a polynucleotide encoding said recombinase.
 10. The method ofclaim 9, wherein said recombinase is flippase (FLP).
 11. The method ofclaim 10, wherein said FLP has been synthesized using maize preferredcodons.
 12. The method of claim 7, wherein said first selectable markerprotein-coding sequence encodes a protein selected from the groupconsisting of a hygromycin phosphotransferase, a sulfonylurea-tolerantacetolactate synthase, and a sulfonylurea-tolerant acetolactate synthasethat has an amino acid sequence comprising SEQ ID NO:63 or SEQ ID NO:64.13. The method of claim 7, wherein the target site comprises a promoteroperably linked to the first selectable marker protein-coding sequence,further wherein the first recombination site is between the promoter andthe first selectable marker protein-coding sequence.
 14. The method ofclaim 7, wherein at least one of said non-identical recombination sitesis selected from the group consisting of FRT1 (SEQ ID NO:50), FRT5 (SEQID NO:51), FRT6 (SEQ ID NO:52), FRT12 (SEQ ID NO:53) and FRT87 (SEQ IDNO:54).